Glycoside-hydrolase enzyme inhibitors

ABSTRACT

This invention relates to inhibitors for glycoside-hydrolases derived from bacteria of the order Actinomycetales, means for their production comprising cultivation of a microorganism of the order Actinomycetales in appropriate nutrient solutions under conditions most favorable to growth and production of the enzyme inhibitor and recovering, as a new product, glycoside-hydrolase enzyme inhibitors, from the culture as well as the use of said enzyme inhibitors in pharmaceutically acceptable therapeutic compositions in the treatment of conditions indicating obesity, diabetes, pre-diabetes, gastritis, gastric ulcer, hyperlipidemia (arteriosclerosis) and the like. The invention also contemplates the provision of methods of inhibiting the reaction of carbohydrates and glycoside-hydrolase enzymes and particularly carbohydrate-splitting glycoside-hydrolase enzymes of the digestive tract by means of conducting said reaction of said carbohydrates and glycoside-hydrolase enzymes in the presence of a glycoside-hydrolase enzyme inhibitor for said glycoside-hydrolase enzyme derived from a strain of microorganism of the order Actinomycetales. The invention further contemplates the provision of methods for the treatment of indications of the group consisting of obesity, adipose, hyperlipidemia (arteriosclerosis), diabetes, pre-diabetes, gastritis, gastric ulcer, duodenal ulcer, and caries induced by the action of glycoside-hydrolase enzymes and carbohydrates, the improvement which comprises employing an enzyme inhibitor for glycoside-hydrolase enzymes produced by a strain of microorganism of the order Actinomycetales.

This is a division of application Ser. No. 213,066 filed Dec. 28, 1971 now U.S. Pat. No. 3,876,766.

BACKGROUND OF THE INVENTION

Carbohydrates are eaten either as simple sugars or as more complex molecules called polysaccharides, which are built up from simple sugar units and of which the starch of potatoes, bread, etc., is a well-known example. The body absorbs carbohydrates from the small intestine in the form of single sugar units, or monosaccharides. Those sugars which are ingested in this form, e.g., glucose in various "energy-giving" preparations and fructose, the sugar of fruit, thus require no further treatment. Sugar molecules bigger than this require enzymic digestion.

The digestion of starch begins in the mouth. Saliva contains an enzyme, amylase, which attacks starch and similar polysaccharides, reducing the size of the molecule. Starch is made up of chains of glucose molecules linked in a particular way, and amylase attacks the links between the glucose units. The enzyme acts at many points in the chain, and the smallest units it produces are molecules of the sugar maltose. Maltose is made up of two glucose units; this sugar is thus a disaccharide. ##SPC1##

Starch also contains branched chains of glucose units, and the amylase of saliva cannot break the chains at the points where branching occurs: ##SPC2##

If the branched-chain structure of starch is thought of as resembling a bush, the enzyme can prune the outer branches until it reaches a fork and then its action stops. Thus, as well as molecules of maltose, amylase produces from starch fragments of the original molecule which are like hard-pruned bushes and which are called "limit dextrins. " Glycogen, the carbohydrate storage material of animal tissues, is also made up of much-branched chains of glucose units.

The digestion of starch or glycogen by salivary amylase probably does get very far, however. The process continues for a while after the food has entered the stomach, but as the acid of the gastric juice penetrates through the food mass the amylase action slows down and stops. The main attack on these polysaccharides takes place in the small intestine by an amylase in pancreatic juice similar to that in saliva.

The dextrins resulting from the "pruning" by amylase are attacked by an enzyme in the small intestine which can break the inter-chain links. A specific enzyme, maltase, also splits each molecule of maltose produced from starch into two molecules of glucose, which are then absorbed and metabolized by the body. Some of the sugars of food are disaccharides; common sugar (sucrose) is made up of a molecule of glucose joined to a molecule of fructose, and lactose (the sugar of milk) is also a disaccharide. These sugars are split into their component monosaccharides by specific enzymes in the intestine.

However, in the treatment of conditions in which there is an indication of obesity, adipose, hyperlipidemia (arteriosclerosis), diabetes, pre-diabetes, gastritis, gastric ulcer, duodenal ulcer, and/or caries, it is necessary that such glycosidehydrolase enzyme be inhibited or suppressed in such a manner that they cannot further catalyze the breakdown thereof, as indicated in the manner above for subsequent utilization by the body and thus further promotion or worsening or the conditiong being treated.

It has been known heretofore that α-amylases can be inhibited by the use of various low molecular substances, such as, for example, salicylic acid and abiscisin [T. Hemberg, J. Larsson, Physiol. Plant. 14, 861 (1961), T. Hemberg, Acta Chem. Scand. 21, 1665 (1967)]. It is further known that there are also higher-molecular substances which are capable of inhibiting the activity of certain amylases non-specifically by physical adsorption [T. Chrzaszcz, J. Janicki, Bioch. Z. 260, 354 (1933) and Bioch. J. 28, 296 (1934)] or by denaturation and precipitation of the enzyme [B. S. Miller, E. Kneen, Arch. Biochem. 15, 251 (1947), D. H. Struhmeyer, M. H. Malin, Biochem. Biophys. Acta 184, 643 (1969)]. It has also been observed that it is possible to elute a substance from wheat by means of distilled water, which lower the dextrifying activity of salivary amylase but has little influence on the activity of pancreatic amylase [E. Kneen, R. M. Sanstedt, Arch., Bioch. 9, 235 (1946)].

It is a disadvantage of these known inhibitors that either the inhibition of the amylase is non-specific or that the inhibiting activity of the inhibitor is slight, especially on pancreatic amylases, as has been shown by our own investigations; that is to say, only at very high ratios of inhibitor: enzyme is almost complete inhibition of the amylases (up to 90 percent and above) attained.

An earlier proposal (U.S. Pat. application Ser. No. 110,482, filed Jan. 28, 1971, now abandoned) relates to amylase inhibitors. In fact, the older proposal shows that by means of aqueous electrolyte solutions, preferably dilute acids, or above all by means of water-alcohol (C₁ -C₃ -alcohols) mixtures, preferably at acid pH values, a highly active inhibitor for pancreatic amylase, which does not show the disadvantages mentioned, can be extracted in high yields from wheat (coarse ground wheat, wheat flour or wheat gluten). The substance thus obtained inhibits pancreatic amylase to the extent of more than 90 percent even at very low inhibitor:enzyme ratios.

THE PRESENT INVENTION

The present invention now relates to inhibitors for glycoside-hydrolases from actinomycetes, and in particular to inhibitors for glycoside-hydrolases of preferably carbohydrate-splitting enzymes of the digestive tract from actinomycetes. These inhibitors are formed by microorganisms of the order Actinomycetales, especially by those of the family of Streptomycetaceae, Thermoactinomycetaces, Micromonospora, Nocardia, and above all those of the family Actinoplanaceae. They are also formed to a particularly high degree by strains of the genera Actinoplanes, Ampullariella, Streptosporangium, Streptomyces, Chainia, Pilimelia, Planomonospora, and Actinobifida.

Accordingly, this invention provides, as a new product, a glycoside-hydrolase inhibitor of microbial origin.

This invention further provides a method for the production of a glycoside-hydrolase inhibitor comprising culturing a microbe of the order Actinomycetales and extracting the inhibitor from the resultant culture.

The preferred microorganisms are preferably of the family Streptomycetaceae or Actinoplanaceae.

A wide variety of microorganisms of the order Actinomycetales have been found to be glycoside-hydrolase enzyme inhibitors and the methods described below can be used to test the microorganisms to determine whether or not the desired glycoside-hydrolase enzyme inhibitor activity is present and the approximate relative value of this activity.

Strains of the order Actinomycetales, especially those of the families Streptomycetaceae and Actinoplanaceae, are isolated in a known manner from samples of soil or strains of those orders bought from culture collections. Culture flasks containing nutrient solutions which permit the growth of these strains are inoculated with inoculum of these strains. For example, the glycerine-glycine nutrient solution according to von Plotho, of composition 2% glycerine, 0.25% glycine, 0.1% NaCl, 0.1% K₂ HPO₄, 0.01% FeSO₄.7 H₂ O, 0.01% MgSO₄.7 H₂ O and 0.1% CaCO₃ can be used. For more rapid growth, it is advisable also to add to such a nutrient solution complex sources of carbon, such as, for example, corn-steep liquor or soya flour or yeast extract or protein hydrolysates, for example NZ-amines, or mixtures of these substances. In these cases, the pH value of the solution must be adjusted. An initial pH of the nutrient solution of between 6.0 and 8.0, especially between 6.5 and 7.5, is preferred.

The glycerine of the nutrientt solution can also be replaced by other sources of carbon, such as, for example, glucose or sucrose or starch or mixtures of these substances. Instead of glycine, it is also possible to use other sources of nitrogen, such as, for example, yeast extract, soya flour, NZ-amines, pharmamedia and others. The concentrations of the sources of carbon and nitrogen, and also the concentrations of the salts, can vary within wide limits. FeSO₄, CaCO₃ and MgSO₄ can also be entirely absent. 100-200 ml, for example, of the nutrient solution are introduced into 1 liter Erlenmeyer flasks, sterilized in a known manner and inoculated with the strain to be investigated, and the flask is incubated on shaking machines at 15°-60°C., preferably at 24°-50°C. If the culture shows growth, which generally takes place after 1-10 days, and in most cases after 3-5 days, a sample of, for example 5 ml is taken and the mycelium in this sample is separated off by filtration or centrifugation. 0-100 μ l of the culture broths are employed in the tests described below, and the inhibiting capacity per ml is calculated.

The mycelia are extracted twice with 5 volumes (relative to the volume of mycelium) of acetone at a time, and subsequently 1 × 5 volumes of diethyl ether, the extracted mycelium residue is dried in vacuo at 20° and the resulting dry mycelium powder is extracted with 4-8 parts by weight of dimethyl sulphoxide (DMSO).

The two acetone extracts and the ether extract are combined and concentrated almost to dryness in vacuo. The residue from these extracts is taken up with the dimethyl sulphoxide (DMSO) extract from the dry powder and 0-100 μl thereof are employed in the tests described below.

AMYLASE TEST

One amylase inhibitor unit (1 AIU) is defined as the amount of inhibitor which inhibits two amylase units to the extent of 50 percent. One amylase unit (AU) is the amount of enzyme which in 1 minute, under the test conditions indicated below, splits 1 μ equivalent of glucoside bonds in starch. The μVal of split bonds are determined colorimetrically as μVal of reducing sugar formed, by means of dinitrosalicylic acid, and are specified as μVal of maltose equivalents by means of a maltose calibration curve. To carry out the test, 0.1 ml of amylase solution (20-22 AU/ml) are treated with 0-400 ug of inhibitor or 0-100 μl of the culture solution or mycelium extracts to be tested, in 0.4 ml of 0.02 M sodium glycerophosphate buffer/0.001 M CaCl₂ pH 6.9, and the mixture is equilibrated in a waterbath at 35°C. for about 10-20 minutes. It is then incubated for 5 minutes at 35°C. with 0.5 ml of a 1% strength starch solution (soluble starch of Messrs. Merck, Darmstadt, No. 1252) which has been pre-warmed to 35°C., and is subsequently treated with 1 ml of dinitrosalicylic acid reagent (according to P. Bernfeld in Colowick-Kaplan, Meth. Enzymol., volume 1, page 149). To develop the color, the batch is heated for 5 minutes on a boiling waterbath and then cooled and treated with 10 ml of distilled water. The extinction at 540 nm is measured against an appropriately mixed blank, without amylase. For evaluation, the amylase activity which is still active after addition of the inhibitor is read off from a previously recorded amylase calibration curve, and the percentage inhibition of the amylase employed is calculated therefrom. The percentage inhibition is plotted as a function of the quotient: ##EQU1## (+ relative to solids, ++ AU in the non-inhibited mix of the same series), and the 50 percent inhibition point is read off from the curve and converted to AIU/mg of inhibitor.

SACCHARASE TEST

One saccharase inhibitor unit (SIU) is defined as the amount of inhibitor which inhibits two saccharase units to the extent of 50 percent. One saccharase unit (SU) is the amount of enzyme which in one minute, under the test conditions indicated below, splits 1 μmol of sucrose into glucose and fructose. The μmol of split sucrose are determined colorimetrically as the glucose and fructose formed, by means of dinitrosalicylic acid, and calculated by means of a glucose/fructose calibration curve.

To carry out the test, 0.1 ml of saccharase solution comprising a solubilized saccharase from intestinal mucous membrane of pigs, [according to B. Borgstrom, A. Dahlquist, Acta Chem. Scand. 12, (1958) page 1997] in the amount of 0.3-0.4 SU/ml are mixed with 0-400 μg of inhibitor or 0-50 μl of the culture solution of the mycelium extract which is to be investigated, in 0.1 ml of a 0.1 M Na maleate buffer of pH 6.0, and the mixture is equilibrated for about 10-20 minutes in a waterbath at 35°C. It is then incubated for 60 minutes at 35+C. with 0.2 ml of an 0.056 M sucrose solution (sucrose: Messrs. Merck, Darmstadt, No. 7652) which has been pre-warmed to 35°C., and is subsequently treated with 0.5 ml of dinitrosalicylic acid reagent (according to P. Bernfeld in Colowick-Kaplan. Meth. Enzymol., Volume 1, page 149). To develop the color, the mix is heated for 5 minutes on a boiling waterbath and then cooled and treated with 5 ml of distilled water. The extinction at 540 nm is measured against an appropriate blank value without saccharase.

For evaluation, the saccharase units which are still active after addition of inhibitor are determined from the calibration curve and the percentage inhibition of the saccharase employed is calculated therefrom. The percentage inhibition is plotted as a function of the quotient: ##EQU2## (+ relative to solids, ++ SU in non-inhibited mix of the same series), and the 50 percent inhibition point is read off from the curve and converted to SIU/mg of inhibitor.

MALTASE TEST

One maltase inhibitor unit (1 MIU) is defined as the amount of inhibitor which inhibits two maltase units to the extent of 50 percent. One maltese unit (MU) is the amount of enzyme which in 1 minute, under the test conditions indicated below, splits 1 μ equivalent of glucosidic bond in the p-nitrophenyl-α-D-glucopyranoside. The μVal of split bonds are determined photometrically as μVal of p-nitrophenolate.

To carry out the test, 0.05 ml of maltese solution comprising a solubilized maltase from intestinal mucous membrane of pigs [according to B. Borgstrom, A. Dahlquist, Acta Chem. Scand. 12, (1958), page 1997], or maltese in the form of human pancreatic juice lyophilizate, (0.09-0.12 MU/ml) are equilibrated with 0-400 μg of inhibitor or 0-20 μl of the culture solution to be investigated or of the mycelium extract in 0.05 ml of 0.1 M sodium maleate buffer at pH 6.0 for about 10-20 minutes in a waterbath at 35°C. The mixture is then incubated for 30 minutes at 35°C. with 0.1 ml of an 0.4 percent strength solution of p-nitrophenyl-α-D-glucyopyranoside (Messrs. Serva, Heidelberg, No. 30,716) in 0.1 M Na maleate buffer at pH 6, which has been pre-warmed to 35°C., and the reaction is subsequently stopped by adding 2 ml of 0.565 M tris-buffer of pH 7.6. The extinction at 403 nm is immediately measured against an appropriately mixed blank without maltase.

For evaluation, the maltase units still active after addition of inhibitor are calculated on the basis of a molar extinction coefficient of E₄₀₃ = 13.2 × 10³ for the p-nitrophenolate anion at pH 7.6, and from the maltase units still active the percentage inhibition of the maltase employed is calculated. The percentage inhibition is plotted as a function of the quotient: ##EQU3## (+ relative to solids, ++ MU in the non-inhibited mix) and the 50 percent inhibition point is read off from the curve and converted to MIU/mg of inhibitor.

Since, in this simple routine test, an artificial substrate and not the actual substrate of maltase (maltose) is used, the preparations are additionally tested for their maltase-inhibiting activity in a more involved maltase test described by Dahlquist (Enzyme. biol. clin. 11, 52/1970). Herein, the glucose produced during the action of maltase on maltose is measured enzymatically by means of colorimetry, using glucose oxidase, peroxidase and dianisidine. All the maltase inhibitors described here also inhibit in this test.

A whole series of strains of various families and genera of the order Actinomycetales was tested in accordance with the method described above. In doing so, distinct, though at times weak, glycoside-hydrolase-inhibiting activities were found in various families and genera. The strains of the family of the Streptomycetaceae and especially of the family of the Actinoplanaceae proved most advantageous as regards yield.

Within these families, particularly active strains were found in the genera Streptomyces, Actinoplanes, Ampullariella and Streptosporangium. Of the strains tested, those listed below proved particularly active in one or more of the tests indicated.

                                      Table 1                                      __________________________________________________________________________                              Inhibiting Action                                     Strain No.     Name      Amylase Maltase Saccharase                            __________________________________________________________________________     Own Desig-                                                                             Collec-                                                                nation  tion Nos.        C   M   C   M   C   M                                 __________________________________________________________________________     SB 2    CBS    Ampullariella                                                                            +++ +                                                         951.70 regularis                                                       SB 5    CBS    "         +++                                                           952.70                                                                 SB 11   CBS    Actinoplanes      +++ +++                                               955.70 spec.                                                           SB 12   CBS    "         +++                                                           956.70                                                                 SB 18   CBS    "         +++ +++ +   ++  ++  ++                                        957.70                                                                 SB 27   CBS    "         +++                                                           958.70                                                                 SB 46   CBS    "         +++                                                           959.70                                                                 SE 5    CBS    Actinoplanes      ++  ++                                                960.70 spec.                                                           SE 21   CBS    Ampullariella                                                                            +++                                                           953.70 regularis                                                       SE 39   CBS    "         ++ +                                                          954.70                                                                 SE 50   CBS    Actinoplanes                                                                             +++ ++  +++ ++  +++ ++                                        961.70 Spec.                                                           SE 55   CBS    "         ++  +   ++  ++  ++  ++                                        962.70                                                                 SS 26   CBS    Streptospor-          ++                                                963.70 angium album                                                    SS 45   CBS    "spec.                ++                                                964.70                                                                 SS 51   CBS    ""                ++  ++                                                965.70                                                                 SS 53   CBS    "roseum           ++  ++                                                966.70                                                                 St 19   ATCC   Streptomyces                                                                             ++                                                            3319   flaveolus                                                       St 50   CBS    Streptomyces              +                                             693.69 heimii                                                          St 67   CBS    Streptomyces      ++      ++                                            432.59 tendae                                                          St 45   CBS    Streptomyces      +                                                     434.51 aureofaciens                                                    RT 36   CBS    Chainia           +                                                     228.65 rubra                                                           RT 33   CBS    Chainia           +                                                     295.66 poonensis                                                       ST 12   NRRL   Streptomyces                                                                             +++     +                                                     B-2286 murinus                                                         ST 51   CBS    Streptomyces                                                                             ++                                                            498.68 fradiae                                                         ST 3    NRRL   Streptomyces                                                                             ++                                                            2580   chrysomallus                                                    ST 1    ATCC   "         ++                                                            11523                                                                  SS 55   CBS    Streptospo-   ++                                                        624.71 rangium roseum                                                  SS 59   "      "amethystogenes                                                                              ++                                                        623.71                                                                 SS 62   "      "roseum       ++                                                        625.71                                                                 AT 8    KCC-A  "viridalbum   ++                                                        0027                                                                   AT 11   KCC-A  "album        ++                                                        0025                                                                   AT 13   CBS    Ampullariella                                                                            +++                                                           19064  campanulata                                                     AT 14   CBS    Ampullariella                                                                            +++                                                           19364  regularis                                                       SE 89   CBS    Ampullariella +++                                                       619.71 spec.                                                           SE 100  CBS    Planomono-                                                                               ++  ++                                                        622.71 spora spec.                                                     AT 4    CBS    Ampullariella     +       ++                                            191.64 digitata                                                        At 9    KCC-A  Streptospo-           +   ++  + +                                       0028   rangium vulgare                                                 AT 10   ATCC   "indianensis              ++  ++                                        19190                                                                  SE 103  CBS    Actinoplanes                  ++                                        616.71 spec.                                                           HN 6    CBS    Actinobifida                  ++                                        602.71 chromogena                                                      HN 2    CBS    ""                            +++                                       601.71                                                                 AT 2    CBS    Actinoplanes      +                                                     367.66 utahensis                                                       SE 101  CBS    Planomonospora        +                                                 621.71 parontospora                                                    SK 2    CBS    Pilimelia spec.               ++                                        620.71                                                                 SE 82   CBS    Actinoplanes                                                                             +++ +++ ++  ++  ++  ++                                        615.71 spec.                                                           SA 28   CBS    Ampullariella     +   +                                                 617.71 digitata                                                        SA 8    CBS    Actinoplanes  +++                                                       611.71 spec.                                                           AT 7    ATCC   Streptosporan-                                                                               ++                                                        12428  gium roseum                                                     SE 45   CBS    Ampullariella                 +                                         618.71 regularis                                                       __________________________________________________________________________      ATCC = American Type Culture Collection                                        CBS = Centraalbureau voor Schimmelcultures, Baarn                              NRRL = Culture Collection Unit, Fermentation Section, Norther Utilization      Research Branch, Peoria, Illinois, USA                                         KCC = Culture Collection, Research Division, Kaken Chemical Co., Ltd.,         Tokyo, Japan                                                                   C = Culture solution                                                           M = Mycelium extract                                                           + = distinctly active                                                          ++ = strongly active                                                           +++ = very strongly active                                               

                                      Table 2                                      __________________________________________________________________________                     Streptosporangium album                                                                        Streptosporangium spec.                                                                        Streptosporangium spec.                        SS 26           SS 45           SS 51                          __________________________________________________________________________     Date of isolation                                                                              August 2, 1967  November 8, 1968                                                                               November 9, 1968               Method          soil smear, plate                                                                              soil smear, plate                              Origin          Rhon, Heidelstein, turf                                                                        Kenya, near Ruiru,                                                                             Kenya, near Ruiru,                             soil between basalt                                                                            coffee plantation,                                                                             coffee plantation,                             blocks, pH 3.9  pH 5.4          pH 5.4                                                         white--cream--yellowish                                                                        white--cream--yellowish        Mycelium   AM   white           or pink         or pink                        SM              0.5-1.2 μ, septate                                                                          0.4-1.2 μ    0.4-1.2 μ, septate          Shape of Sporangia                                                                             spherical, in part                                                                             spherical, smooth                                                                              spherical, smooth, some                        wrinkled and deformed           also wrinkled and irre-                                                        gular, shriveled forms         Size of Sporangia                                                                              3-7 μ φ  3-11 μ φ 3-11 μ φ                Shape of Spores ± spherical to elongated                                                                    mostly ± oval                                                                               mostly ± oval, in                                                           parts                                                                          spherical                      Size of Spores  0.6-0.9 × 0.7-1.1 × 0.7-1.0 ×                                0.6-1.3 μ    1.0-1.7 μ    0.8-1.7 μ                   Flagellation    -               -               -                              Arrangement of  spiral chains   spiral chains   spiral chains                  Spores in Sporangium                                                           Conidia, Substrate                                                             spores and the like                                                                            PEH -           PEH -           PEH -                          Melanine        CPC - -         CPC - -         CPC - -                                        Ty -            Ty -            Ty -                                           Gel -           Gel -           Gel -                          Nitrate reduction                                                                              + (weak)        -               -                              Gelatine liquefaction                                                                          +               +               +                              Milk peptonization                                                                             +                                                              Growth at                                                                              20°C.                                                                           +               +               +                                      26°C.                                                                           ++              ++              ++                                     32°C.                                                                           ++              +               +(+)                                   37°C.                                                                           -               +               +                                      42°C.                                                                           -               -               -                                      2%      + slight growth ++              ++                             NaCl    3%      -               ++              ++                             toleration                                                                             4%      -               +               +                                      7%      -               -               -                              Starch hydrolysis                                                                              -               +               +                              __________________________________________________________________________                     Streptosporangium roseum                                                                       Actinoplanes spec.                                             SS 53           SE 5            SE 55                          __________________________________________________________________________     Date of isolation                                                                              November 9, 1968                                                                               December 16, 1969                                                                              December 31, 1969              Method          soil smear, plate                                                                              pollen          pollen                         Origin          Kenya, near Ruiru coffee                                                                       Kenya, near Ruiru                                                                              Kenya, Njoro, Eldoret                          plantation, pH 5.7                                                                             coffee plantation,                                                                             Experimental Station,                                          pH 5.6          pH 5.1                         Mycelium   AM   pink            0.4-1.3 μ    0.3-1.3 μ, septate          SM              0.4-1.2 μ                                                   Shape of Sporangia                                                                             spherical, in part              irregular                                      slightly wrinkled                                              Size of Sporangia                                                                              3-10 μ φ                 about 4-12 μ                Shape of Spores mostly ± oval                ± spherical                 Size of Spores  0.7-1.1 ×                                                                1.0-1.9 μ                    Ca. 1 μ                     Flagellation    -                                                              Arrangement of Spores                                                                          spiral chains                   coiled chains; spore           in Sporangium                                   chains also in places                                                          running parallel and                                                           straight                       Conidia, Substrate              on CPC: substrate                              spores and the like             spores, mostly spheri-                                                         cal (±), up to approx.                                                      2 μ φ individual or                                                     several together, ter-                                                         minal or intercalar                                            PEH -                                                          Melanine        CPC -           CPC -           CPC +                                          Ty -            Ty -            Ty +                                           Gel -                                                          Nitrate reduction                                                                              +                                                              Gelatine        +                                                              liquefaction                                                                   Milk peptonization                                                                             +               -               -                              Growth at                                                                              20°C.                             ++                                    26°C.                                                                           ++                              ++                                     32°C.                                                                           ++                              ++                                     37°C.                                                                           +                               ++                                     42°C.                                                                           -                               -                                      2%      +(+)                                                           NaCl    3%      +                                                              toleration                                                                             4%      -                                                                      7%      -                                                              Starch hydrolysis                                                                              +                               +                              __________________________________________________________________________                     Ampullariella regularis                                                                        Ampullariella regularis                                                                        Ampullariella regularis                        SB 2            SB 5            SE 21                          __________________________________________________________________________     Date of isolation                                                                              July 2, 1966    July 3, 1966    December 17, 1969              Method          pollen          pollen          pollen                         Origin          Neuhof, Fulda district,                                                                        Neuhof, Fulda district,                                                                        Kenya, near Ruiru                              soil under rotted straw,                                                                       soil under rotted straw,                                                                       coffee plantation,                             edge of field, pH 5.7                                                                          edge of field, pH 5.7                                                                          pH 5.3                         Mycelium        0.3-1 μ, broad                                                                              0.3-1 μ      0.3-1 μ                     Shape of Sporangia                                                                             bottle-shaped,  bottle-shaped,  bottle-shaped,                                 cylindrical, "money-                                                                           cylindrical, "money-                                                                           cylindrical, "money-                           pouch-shaped"   pouch-shaped"   pouch-shaped"                  Size of Sporangia                                                                              4-10 ×    3.5 -7 ×  3.5-9 ×                                  7-18 μ       6-14 μ       6-13 μ                      Shape of Spores small rods      small rods      small rods                     Size of Spores  0.5-0.7 × 0.5-0.7 × 0.5-0.7 ×                                1.5-2.2 μ    1.5-2.2 μ    1.5-2.2 μ                   Flagellation    lophotrichous   lophotrichous   lophotrichous                  Arrangement of Spores                                                                          linear parallel chains                                                                         linear parallel chains                                                                         linear parallel chains         in Sporangium                                                                  Conidia, Substrate                                                             spores and the like                                                                            CPC -           CPC -           CPC +                          Melanine        Ty -            Ty -            Ty +                                           Gel -           Gel -                                          Nitrate reduction                                                                              +               +                                              Gelatine liquefaction                                                                          +               +                                              Milk peptonization                                                                             +               +               +                              Starch-hydrolysis                                                                              +               +               +                              Growth at                                                                              20°C.                                                                           ++              ++              ++                                     26°C.                                                                           ++              ++              ++                                     32° C.                                                                          ++              ++              ++                                     37°C.                                                                           -               -               ++                                     42°C.                                                                           -               -               -                              __________________________________________________________________________                     Ampullariella regularis                                                                        Actinoplanes spec.                                                                             Actinoplanes spec.                             SE 39           SB 11           SB 12                          __________________________________________________________________________     Date of isolation                                                                              December 22, 1966                                                                              November 9, 1966                                                                               November 9, 1966               Method          pollen          pollen          pollen                         Origin          Kenya, near Ruiru                                                                              Eschwege district,                                                                             Arfurt, Oberlahn                                                               district,                                      coffee plantation,                                                                             near Frankershausen                                                                            soil from sunny position                       pH 5.6          "Hielocher", pH 7.7                                                                            on rock, pH 7.3                Mycelium        0.3-1 μ      0.3-1.3 μ, septate                                                                          0.3-1.2 μ                   Shape of Sporangia                                                                             bottle-shaped,                  ± spherical with                            cylindrical, "money-            wrinkled surface, in                           pouch-shaped"                   part irregular                 Size of Sporangia                                                                              3-10 ×                                                                   5-16 μ                       3.5-12 μ                    Shape of Spores small rods                      ± spherical                 Size of Spores  0.5-0.7 ×                 approx.                                        1.5-2.2 μ                    1-1.3 μ                     Flagellation    lophotrichous                   mobile spores                  Arrangement of Spores                                                                          linear parallel chains          coiled-up chains               in Sporangium                                                                  Conidia, Substrate              on CPC broad hyphen                            spores and the like             (approx. 1.3 μ),                                                            septate at intervals                                                           of approx. 2.5-4 μ                                                          optically dense,                                                               permanent cells?                               Melanine        CPC +           CPC -           CPC -                                          Ty +                +           Ty - +                                                         Gel +           Gel +                          Nitrate reduction               +               -                              Gelatine liquefaction           +               +                              Milk peptonization                                                                             -               +               +                              Starch-hydrolysis                                                              Growth at                                                                              20°C.                                                                           ++              ++              ++                                     26°C.                                                                           ++              ++              ++                                     32°C.                                                                           ++              ++              ++                                     37°C.                                                                           ++              +               ++                             42°C.                                                                           -       -               -                                              __________________________________________________________________________                  Actinoplanes Actinoplanes Actinoplanes                                                                              Actinoplanes                              spec.        spec.        spec.      spec.                                     SB 18        SB 27        SB 46      SE 50                        __________________________________________________________________________     Date of isolation                                                                           December 4, 1966                                                                            December 8, 1966                                                                            March 3, 1967                                                                             December 22, 1969            Method       pollen       pollen       pollen     pollen                       Origin       Hattenheim,  Kuhkoph, Nature                                                                             Neuhof, Fulda                                                                             Kenya, near Ruiru,                        Rheingau, potato                                                                            Reserve, Altrhein,                                                                          district, turf                                                                            coffee plantation,                        field in the riverside wood,                                                                             soil, edge of                                                                             pH 6.2                                    "Sandaue", pH 8.0                                                                           under willows,                                                                              path, pH 6.1                                                      pH 7.6                                               Mycelium     0.3-1.3 μ 0.2-1.1 μ 0.3-1.4 μ                                                                              0.4-1.3 μ                              septate                   septate                                 Shape of Sporangia                                                                          irregular,   ± club-shaped or                                                                         irregular, irregular, humped,                        wrinkled, humped                                                                            oval to cylindri-                                                                           folded, humped                                                                            wrinkled                                               cal, in part irre-                                                             gular, surface                                                                 wrinkled                                             Size of Sporangia                                                                           6-16 μ    4-16 μ    6-20 μ  4-13 μ                    Shape of Spores                                                                             ± spherical, in                                                                          ± spherical                                                                              ± spherical                                                                            ± spherical                            part with nose-                                                                like protuberance                                                 Size of Spores                                                                              1-1.7 μ   1-1.3 μ   approx. 1.2-2 μ                                                                        approx. 1 μ               Flagellation bundles of flagellae                                                                        mobile spores                                        Arrangement of                                                                              coiled chains                                                                               slightly coiled,                                                                            coiled chains,                                                                            coiled chains,               Spores in                 in part parallel                                                                            in part parallel                                                                          parallel in places           Sporangium                and straight                                                                   chains                                               Conidia, Substrate                     on CPC substrate                        spores and the like                    spores, up to 3 μ                                                           φ, singly or inter-                                                        calar to give several                                                          in a row                                Melanine     CPC -        CPC -        CPC -      CPC +                                     Ty - +       Ty - -       Ty - -     Ty + +                                    Gel +        Gel -        Gel -                                   Nitrate reduction                                                                           -            +            -          -                            Gelatine liquefaction                                                                       +            +            +                                       Milk peptonization                                                                          +            +            +          +                            Starch-hydrolysis                                                                           +                                    +                            Growth at                                                                             20°C.                                                                         ++           ++           ++         ++                                  26°C.                                                                         ++           ++           ++         ++                                  32°C.                                                                         ++           ++           ++         ++                                  37°C.                                                                         +            +            -          ++                                  42°C.                                                                         -            -            -          -                            __________________________________________________________________________                     Actinoplanes    Actinoplanes    Actinoplanes                                   spec.           spec.           spec.                                          SA 8            SE 82           SE 103                         __________________________________________________________________________     Date of isolation                                                                              June 26, 1966   February 26, 1971                                                                              March 2, 1971                  Method          pollen          pollen          pollen                         Origin          Marburg, compost                                                                               Ceylon, earth,  Corsica, near Holy                             earth, botanic  rubber plantation                                                                              Trinity, from earth                            garden                          under cork-oaks                Mycelium        0.3-1.3 μ    0.3-1.3 μ                                   Shape of Sporangia                                                                             irregular       irregular       irregular                      Size of Sporangia                                                                              7-15 × 9-18 μ                                                                         5-14 μ       about 5-13 μ                Shape of spores ± spherical  ± spherical  ± spherical                 Size of spores  about 1.5 μ  about 1-1.4 μ                                                                               about 1 μ                   Flagellation    mobile                          rapidly mobile                 Arrangement of  coiled chains   coiled chains                                  Spores in                                                                      Sporangium                                                                                     Ty +                                                           Melanine        Gel + +                                                                        CPC -                                                          Nitrate reduction                                                                              + (very low)                                                   Gelatine liquefaction                                                                          +                                                              Milk peptonization                                                                             +                                                              __________________________________________________________________________                     Streptosporangium                                                                              Streptosporangium                                                                              Streptosporangium                              roseum          amethystogenes  roseum                                         SS 55           SS 59           SS 62                          __________________________________________________________________________     Isolation date  November 10, 1968                                                                              November 10, 1968                                                                              November 10, 1968              Method          soil smear, plate                                                                              soil smear, plate                                                                              soil smear, plate              Origin          Kenya, near Ruiru,                                                                             Kenya, near Njoro                                                                              Kenya, near Njoro,                             earth from coffee                                                                              from field      from field                                     field                                                          Mycelium        0.4-1.2 μ                    0.4-1.2 μ                   Shape of sporangia                                                                             spherical       spherical       spherical                      Size of sporangia                                                                              3-10 μ       3-15 μ       3-10 μ                      Shape of spores mostly oval                     mostly oval                    Size of spores  about 0.8 ×1-1.2 μ     about 0.8-1.1 ×1-                                                        1.5 μ                       Melanine        -               -               -                              Nitrate reduction                                                                              ++              -               ++                             Gelatine liquefaction                                                                          +               +               +                              Milk peptonization                                                                             +               +               +                              Starch hydrolysis                                                                              +               +               +                              Growth at                                                                              37°C.                                                                           +               +               +                                      42°C.                                                                           -               -               -                                      2%                      +                                              NaCl    3%      +               -               +                              toleration                                                                             4%      -                               -                              __________________________________________________________________________                     Ampullariella   Ampullariella   Ampullariella                                  digitata        regularis       spec.                                          SA 28           SE 45           SE 89                          __________________________________________________________________________     Isolation date  November 10, 1967                                                                              December 23, 1969                                                                               February 27, 1971             Method          fungus mycelium pollen          pollen                         Origin          Amonau Krs. Marburg,                                                                           Kenya, near Ruiru,                                                                             Ceylon, earth                                  earth from stubble-                                                                            earth from coffee                                              field           field                                          Mycelium        0.3-1 μ                                                     Shape of sporangia                                                                             very narrow and rela-                                                                          bottle-shaped,  ± cylindrical, fre-                         tively long, distally                                                                          cylindrical, often                                                                             quently also broader                           and in part also                                                                               longitudinally narrow                                                                          than long, often not                           laterally irregular,            flat at the distal end,                        in part fingered,               tubercular, in part                            coil-like or finger-            also irregular and                                                             folded                                         shaped                          a few fingered                 Size of sporangia                                                                              2-7 × 4-13 μ                                                                          4-10 × 6-17 μ                                                                         5-12 × 6-16 μ         Shape of sporangia                                                                             small rod-shaped                                                                               small rods      small rod-shaped               Arrangement of  arranged in parallel                                                                           in parallel                                    spores in sporangia                                                                            rows            straight rows                                                  Ty -            Ty +                                           Melanine        Gel - +                                                                        CPC +                                                          Nitrate reduction                                                                              +                                                              Gelatine liquefaction                                                                          +                                                              Milk peptonization                                                                             +               +                                              __________________________________________________________________________                     Planomonospora  Planomonospora  Pilimelia spec.                                spec.           parontospora    KS.sup.2                                       SE 100          SE 101                                         __________________________________________________________________________     Isolation date  March 1, 1971   February 27, 1971                                                                              January 28, 1968               Method          pollen          pollen          pollen                         Origin          Corsica, northern                                                                              Ceylon, lawn earth                                                                             Marburg, garden earth,                         Algeria, earth,                 Schulerpark                                    tree nursery                                                   Mycelium                                        0.3-0.7 μ                   Shape of sporangia              longitudinally narrow,                                                                         spherical, pear-shaped,                                        stand compactly by each                                                                        oval, with a columella,                                        other in parallel double                                                                       which projects, as a                                           rows in the air mycelium,                                                                      continuation of the                                            arcuately bent air-hyphens                                                                     sporangiophor, up to                                           on the convex side di-                                                                         about 1/3 of the                                               rectly joined to their                                                                         sporangia - φ or                                                           further                                                        base            into the sporangium            Size of sporangia               about 1-1.3 ×                                                                            about 7-15 μ                                                3-4.5 μ                                     Shape of spores                                 small rods often                                                               slightly warped                Size of spores                                  0.35-0.45 ×                                                              0.8-1.5 μ                   Flagellation                                    mobile flagellar                                                               fascides                       Arrangement of                                  lateral (and also sub-         spores in                                       polar), spore arrange-         sporangium                                      ment in chains, which                                                          grow tuft-like from                                                            the columella                  Melanine                                        Ty +                           Nitrate reduction                               +                              Gelatine liquefaction                           +                              Milk peptonization                              +                              Growth at                                                                              32°C.                            +                                      37°C.                            -                              __________________________________________________________________________

                                      Table 3                                      __________________________________________________________________________      G = growth           SM = substrate mycelium                                                                             SP = soluble pigment                Spg = sporangium       C = colony shape    AM = aerial mycelium                __________________________________________________________________________                 SB 2             SB 5             SB 11                            __________________________________________________________________________               (1)                                                                               G.  very good   G.   good        G.   good                                  (2)                                                                               SM. orange      SM.  yellow-orange                                                                              SM.  beautiful orange,           Casamino-peptone-                                  bleaching                             (3)                                                                               SP. agar yellowish                                                                             SP.  agar yellowish                                                                             SP.  agar yellowish              Czapek agar                                                                              (4)                                                                               Spg.                                                                               +++; rimy   Spg. -; originally +                                                                            Spg. -                           (CPC)     (5)                                                                               C.  flat and smooth                                                                            C.   heavily veined                                                                             C.   with coiled ridges                      G.   very good   G.   good to very good                                                                          G.   good                        Peptone-    SM.  orange, rimy                                                                               SM.  orange-red  SM.  orange, bleaching                       SP.  -           SP.  agar yellowish                                                                             SP.  agar golden yellow          Czapek agar Spg. ++          Spg. -           Spg. -                                       C.   flat, in part                                                 (PC)             humped                                                                    G.   moderate    G.   moderate    G.   good                        Czapek agar SM.  orange      SM.  pale orange SM.  brownish orange             (Cz)        SP.  agar pale ochre                                                                            SP.  agar slightly                                                                              SP.  -                                                             yellowish                                                Spg. ++          Spg. -           Spg. -                                       G.   good        G.   very good   G.   very good                   Milk agar   SM.  brownish orange                                                                            SM.  brownish orange                                                                            SM.  brownish orange,                                                               turns pale brown            (Ca)        SP.  agar brownish                                                                              SP.  agar golden brown                                                                          SP.  agar yellow brown                       Spg. -; casein   Spg. -; casein   Spg. -; casein                                    peptonized       peptonized       peptonized                              G.   moderate-good                                                                              G.   slight      G.   bulky                                   SM.  reddish brown                                                                              SM.  brown       SM.  pale orange                 Tyrosine agar                                                                              SP.  agar reddish                                                                               SP.  agar brown  SP.  -                                            brown, slight    solution of      Tyrosine crystals           (Ty)             solution of      crystals -       not dissolved                                crystals                                                                  Spg. +; rimy     Spg. -                                                        G.   good        G.   good        G.   good                        Oat-Yeast agar                                                                             SM.  brownish orange                                                                            SM.  brownish orange                                                                            SM.  ochre-orange                            SP.  -           SP.  -           SP.  -                           (OY)        Spg. +++         Spg. +           Spg. -                                                                         G.   bulky                                                                     SM.  colorless to pale                                                              ochre                       Starch agar                                   SP.  -                                                                         Spg. -                           __________________________________________________________________________                 SB 12            SB 18            SB 27                            __________________________________________________________________________                 G.   very good   G.   very good   G.   good to very good           Casamino-Peptone-                                                                          SM.  orange-brown                                                                               SM.  strong red-brown                                                                           SM.  luminous orange,                                                               later brown orange          Czapek agar SP.  agar yellowish-                                                                            SP.  agar brown  SP.  agar yellow-brown                            brownish                                                      (CPC)       Spg. +           Spg. +(+)        Spg. +                                       C.   flat with radial                                                                           C.   flat, radial                                                                               C.   flat, bulging in                             grooves, bulging grooves and      the middle, locally                          in the middle    wrinkles         split open                              G.   very good   G.   very good   G.   good to very good           Peptone-Czapek agar                                                                        SM.  brown red   SM.  brown       SM.  ochre orange                            SP.  agar golden brown                                                                          SP.  brown       SP.  agar yellowish              (PC)        Spg. -           Spg. ++          Spg. -                                       G.   good        G.   good        G.   good                                    SM.  orange to   SM.  red brown   SM.  red brown                                    brownish orange                                               Czapek agar SP.  agar fluoresces                                                                            SP.  agar reddish                                                                               SP.  agar slightly                                greenish to      brown            ochre                       (Cz)             yellow                                                                    Spg. -           Spg. ++; rimy    Spg. -                                       G.   very good   G.   very good   G.   very good                   Milk agar   SM.  brown orange                                                                               SM.  brown orange                                                                               SM.  brownish orange                         SP.  agar golden brown                                                                          SP.  agar golden brown                                                                          SP.  agar golden brown           (Ca)        Spg. -;          Spg. ++,         Spg. +,                                           casein peptonized                                                                               not always;      not always;                                                   casein peptonized                                                                               casein peptonized                       G.   moderate    G.   moderate - good                                                                            G.   moderate - good             Tyrosine agar                                                                              SM.  brown       SM.  reddish orange brown                                                                       SM.  ochre brown                             SP.  agar brown; SP.  agar orange brown;                                                                         SP.  -                           (Ty)             crystal solution crystal solution ++                                                                             crystal solution                             ++                                ++                                      Spg. -           Spg. -           Spg. -                                       G.   good        G.   good        G.   good                        Oat-Yeast agar                                                                             SM.  pale orange SM.  red brown   SM.  orange brown                            SP.  -           SP.  red brown   SP.  -                           (OY)        Spg. +           Spg. ++          Spg. -                                       G.   bulky to good                                                                              G.   bulky to good                                                                              G.   bulky to good               Starch-agar SM.  pale brownish orange                                                                       SM.  pale brownish orange                                                                       SM.  pale brownish orange                    SP.  -           SP.  -           SP.  -                                       Spg. +           Spg. ++          Spg. ++                          __________________________________________________________________________             SB 46         SE 21        SE 39         SE 50                         __________________________________________________________________________             G.  good      G.  good     G.  good      G.  very good                 Casamino-                                                                              SM. orange, later                                                                            SM. orange brown                                                                            SM. red brown SM. brown                                 brownish orange                                                    peptone-                                                                               SP. agar pale SP. agar yellow                                                                             SP. agar golden                                                                              SP. agar brown to                         yellowish     brown        brown - red   yellowish                 Czapek agar                            brown         brown                     (CPC)   Spg.                                                                               +         Spg.                                                                               +; some  Spg.                                                                               ++; rimy  Spg.                                                                               -                                 C.  flat, with radial                                                              grooves                                                                    G.  good      G.  very good                                                                               G.  very good G.  very good                 Peptone-                                                                               SM. orange    SM. dark brown                                                                              SM. reddish dark                                                                             SM. brown                                                            brown                                   Czapek agar                                                                            SP. agar golden                                                                              SP. brown    SP. dark brown                              (PC)        yellow                                                                     Spg.                                                                               -         Spg.                                                                               +        Spg.                                                                               ++ frost-like                                                                  on the SM                                       G.  good      G.  good     G.  good      G.  very good                         SM. orange brown                                                                             SM. orange   SM. red-brown SM. red-brown                         SP. agar ochre-                                                                              SP. - to pale                                                                               SP. red       SP. gold brown                            colored       yellowish                                                    Spg.                                                                               -         Spg.                                                                               +++ frost-like                                                                          Spg.                                                                               +                                                                 on the SM                                                    G.  very good G.  good     G.  good      G.  good                      Milk agar                                                                              SM. orange    SM. brown-orange                                                                            SM. brown-orange                                                                             SM. orange brown                      SP. agar golden-                                                                             SP. brown    SP. dark brown                                                                               SP. dark brown                (Ca)        colored                                                                    Spg.                                                                               -; casein Spg.                                                                               - casein Spg.                                                                               - casein not                                                                             Spg.                                                                               - casein                              peptonized    peptonized   peptonized    peptonized                        G.  good      G.  bulky    G.  bulky     G.  bulky                     Tyrosine agar                                                                          SM. brown     SM. dark brown                                                                              SM. dark brown                                                                               SM. dark, like the                                                                 agar                      (Ty)    SP. agar golden                                                                              SP. dark brown                                                                              SP. dark brown                                                                               SP. black-brown                           brown; crystal                                                                 solution ++                                                                Spg.                                                                               -         Spg.                                                                               -        Spg.                                                                               -         Spg.                                                                               -                                                   tyrosine crystals                                                                           tyrosine crystals                                                                            tyrosine crystals                                   not dissolved                                                                               not dissolved not dissolved                     G.  good                                                               Oat-Yeast agar                                                                         SM. orange brown                                                               SP. -                                                                  (OY)    Spg.                                                                               -         Spg.                                                                               ++                     Spg.                                                                               +                         "Emerson"                                        G.  good                      agar E.                                          SM. brown                     (yeast-starch                                    SP. brown                     agar)                              Spg.                                                                               ++        Spg.                                                                               -                                               G.  bulky to good                                                                           G.  bulky to good                                                                            G.  bulky to good             Starch agar           SM. pale brownish                                                                           SM. pale brownish                                                                            SM. pale brownish                                                    orange        orange                                          SP. -        SP. -         starch hydrolysis -                                 Spg.                                                                               +        Spg.                                                                               +         Spg. +                        __________________________________________________________________________                 SE 5             SE 55            SE 103                           __________________________________________________________________________                 G.   good        G.   good        G.   good                                    SM.  orange      SM.  dark red brown                                                                             SM.  orange                      Casamino-   AM.  -           AM.  -                                            peptone-    SP.  - to pale yellowish                                                                        SP.  yellow brown                                                                               SP.  yellowish-brownish          Czapek agar                                   Spg. -                           (CPC)       surface of colony in                                                           inclined test-tube shows                                                       slimy gloss                                                                    G.   good        G.   good        G.   very good                   Oat-Yeast   SM.  orange      SM.  orange brown                                                                               SM.  brown                       agar                         AM.  -                                            (OY)                         SP.  light brown SP.  -                                                                         Spg. ++, frost-like                          G.   good                         G.   very good                               SM.  orange                       SM.  orange                                  AM.  -                                                                         SP.  -                            SP.  -                                       surface of colony                 Spg. ++, frost-like                                                            slimy                                        G.   good        G.   good                                         Milk agar   SM.  orange      SM.  orange brown                                 (Ca)        SP.  - to yellowish                                                                             SP.  brown                                                         Casein not peptonized,                                                                          Casein not peptonized,                                        except merely negli-                                                                            after about 2 months                                          gibly immediately                                                                               slightly peptonized                                           around the mycelium                                                       G.   good        G.   good        G.   very good                   Czapek agar SM.  orange, surface                                                                            SM.  orange      SM.  yellow-orange               (Cz)             damp and shiny                                                            SP.  -           SP.  -           SP.  -                                                                         Spg. -                                       G.   small to bulky                                                                             G.   bulky                                        Tyrosine agar                                                                              SM.  colorless to pale                                                                          SM.  dark brown                                   (Ty)             orange                                                                    SP.  -           SP.  black brown                                                   tyrosine crystals                                                                               tyrosine crystals                                             not dissolved    not dissolved                                Peptone-Czapek               G.   very good                                    Agar                         SM.  dark reddish brown                           (PC)                         SP.  dark brown                                                                G.   bulky to good                                Starch agar                  SM.  pale orange brown                                                         SP.  -                                                                         Spg. +                                            __________________________________________________________________________                 SS 45            SS 51            SS 53                            __________________________________________________________________________                 G.   moderate (to good)                                                                         G.   moderate (to good)                                                                         G.   good                        Casamino-   SM.  reddish brown                                                                              SM.  reddish brown                                                                              SM.  pale light brown            peptone-    AM.  -           AM.  -           AM.  white                       Czapek agar SP.  -           SP.  -           SP.  brownish                    (CPC)            colony flat, 6 mm φ                                                                         colony flat, 6 mm φ                                                                         colony flat, center                          after 9 weeks    after 9 weeks    without AM                              G.   good        G.   good        G.   good to very good           Oat-Yeast   SM.  brown red   SM.  brown red   SM.  purple brown to             agar                                               red brown                   (OY)        AM.  +, white and pink                                                                          AM.  +, white and pink                                                                          AM.  ++, dirty white and                                                            pink                                    SP.  pale yellowish to                                                                          SP.  pale yellowish with                                                                        SP.  purple-red brown                             yellowish-greenish                                                                              greenish tinge                                           Spg. +           Spg. +           Spg. ++                                      G.   good        G.   good        G.   good                        "Emerson"   SM.  red         SM.  brown red   SM.  red brown                   agar        AM.  -           AM.  +, slight, white                                                                           AM.  ++, pink                    (E)         SP.  pale yellowish                                                                             SP.  -to pale yellowish                                                                         SP.  red brown                                    with greenish tinge                                                                             brownish                                                                                   Spg. ++                                      G.   good        G.   good        G.   good                        Milk agar   SM.  pale brownish red                                                                          SM.  pale brownish red                                                                          SM.  pale light brown            (Ca)        AM.  -           AM.  -           AM.  -                                       SP.  agar gold-yellow                                                                           SP.  agar gold-yellow                                                                           SP.  agar yellow brown                            Casein peptonized                                                                               Casein peptonized                                                                               Casein peptonized                       G.   good        G.   good        G.   good                        Yeast-glucose-soil                                                                         SM.  brownish red                                                                               SM.  brown red   SM.  brownish claret             extract agar                                                                               AM.  + thin coating                                                                             AM.  +, white    AM.  ++, pink                    (YGS)       SP.  golden yellow with                                                                         SP.  yellow with greenish                                                                       SP.  reddish yellow                               greenish tinge   tinge            brown                       Manure extract                                                                             G.   moderate (to good)                                                                         G.   moderate (to good)                           agar        AM.  ++, white and pink                                                                         AM.  ++, pink                                     (Ma)        Spg. +           Spg. +                                            __________________________________________________________________________                 SA 8             SE 82            SS 26                            __________________________________________________________________________                 G.   very good   G.   good        G.   good                        Casamino-   SM.  orange      SM.  brown       SM.  pale ochre                  peptone                                       AM.  few, white                  Czapek      SP.  yellowish-brownish                                                                         SP.  brown       SP.  -                           agar        Spg. +                            Spg. -                           (CPC)                                              colony flat with a                                                             few                                                                            radial grooves and                                                             con-                                                                           centric humps               Peptone     G.   very good                                                     Czapek agar SM.  orange                                                        (PC)        SP.  yellowish-brownish                                                        Spg. +                                                                         G.   good        G.   very good   G.   good to bulky               Oat-Yeast   SM.  brown       SM.  orange to brownish                                                                         SM.  colorless                   agar                              orange                                       (OY)                                          AM.  few, white                              SP.  -           SP.  weak yellowish-                                                                            SP.  -                                                             brownish                                                 Spg. +                            Spg. +                                                        G.   very good   G.   bulky                       "Emerson"                    SM.  orange-brown                                                                               SM.  colorless                   agar                                          LM.  +, white                    (E)                          SP.  brown       SP.  -                                       G.   very good                    G.   good                        Milk agar   SM.  orange brown                 SM.  colorless                   (Ca)        SP.  agar gold-brown              LM.  -                                       Spg. +                            SP.  -                                            Casein peptonized                 Casein peptonized           Czapek      G.   good        G.   good                                         agar        SM.  brown-orange                                                                               SM.  red-orange                                   (Cz)        SP.  yellow brown                                                                               SP.  yellowish                                                Spg. -                                                             Tyrosine    G.   bulky                                                         agar        SM.  black                                                         (Ty)        SP.  black                                                                          tyrosine crystals                                                              dissolved                                                                 Spg. -                                                             Yeast-glucose-                                                                             G.   good                         G.   good                        soil extract                                                                               SM.  brown                        SM.  colorless                   agar        SP.  gold-brown                   LM.  +, white                    (YGS)       Spg. +                            SP.  -                           Manure extract                                G.   bulky                       agar                                          SM.  pale brown                  (Ma)                                          LM.  +, white                                                                  Spg. +                           __________________________________________________________________________                 SS 55            SS 59            SS 62                            __________________________________________________________________________     Casamino-   G.   good, colony flat            G.   good, colony flat           peptone-    SM.  light brown                  SM.  pale light brown            Czapek      AM.  white                        AM.  white                       agar (CPC)  SP.  weak pale brown              SP.  -                                       G.   good        G.   good        G.   good                                    SM.  pale light brown                                                                           SM.  dark brown  SM.  pale light brown            Milk        AM.  -           AM.  -           AM.  -                           agar        SP.  -           SP.  yellow brown                                                                               SP.  agar yellow-brown                            Casein peptonized                                                                               violet crystals  Casein peptonized                                             Casein peptonized                                        G.   good        G.   good        G.   good                                    SM.  brown       SM.  dark brown  SM.  brown                       Tyrosine    AM.  white       AM.  -           AM.  few, white                  agar        SP.  -           SP.  brown       SP.  brown                       (Ty)             tyrosine crystals                                                                               tyrosine crystals                                                                               tyrosine crystals                            dissolved        dissolved        dissolved                               G.   good to very good                                                                          G.   good        G.   good to very good           Oat-Yeast   SM.  red-brown   SM.  dark brown  SM.  red brown.                  agar        AM.  dirty pink  AM.  few, white  AM.  dirty pink                  (OY)        SP.  red brown   SP.  brownish    SP.  red brown                                                     many violet crystals                                                      Spg. -                                                        G.   good        G.   good        G.   good                        "Emerson"   SM.  red brown   SM.  dark brown  SM.  red brown                   agar        AM.  pink and white                                                                             AM.  -           AM.  few, thin coating,          (E)                                                in part stronger                                                               and pink                                SP.  red brown   SP.  yellow brown                                                                               SP.  red brown                                                     violet crystals                                          G.   good        G.   good        G.   good                        Yeast-glucose                                                                              SM.  red brown   SM.  dark brown  SM.  dark red                    soil extract                                                                               AM.  pink        AM.  -           AM.  pink                        agar        SP.  yellow-brown                                                                               SP.  yellow brown                                                                               SP.  yellow brown                (YGS)                             violet crystals                              __________________________________________________________________________                 SA 28            SE 45            SE 89                            __________________________________________________________________________                 G.   good to very good                                                                          G.   good        G.   good                        Casamino    SM.  initially red-brown,                                                                       SM.  red-brown   SM.  brownish orange             peptone          later dark brown                                              Czapek agar      to black                                                      (CPC)       SP.  dark olive  SP.  strongly red-brown                                                                         SP.  -                                       Spg. -           Spg. -           Spg. +                                       G.   good to very good                                             Peptone     SM.  initially red-brown,                                          Czapek           later dark brown                                              agar             to black                                                      (PC)        SP.  dark olive                                                                Spg. -                                                                         G.   bulky to good, colonies                                                                    G.   good        G.   good                        Czapek agar      flat                                                          (Cz)        SM.  orange brown                                                                               SM.  brown-red   SM.  dark-brown                              SP.  weak ochre  SP.  brownish-red                                                                               SP.  light yellowish brown                   Spg. +, in part frost-like                                                                      Spg. ++, frost-like                                                                             Spg. ++, frost-like                          G.   very good   G.   good                                         Milk agar   SM.  black brown SM.  orange brown                                 (Ca)        SP.  dark olive to                                                                              SP.  brown                                                         greenish-black                                                            Spg. -           Spg. +                                                             Casein peptonized                                                                               Casein peptonized                                        G.   bulky to good                                                                              G.   bulky                                        Tyrosine    SM.  brown       SM.  brown                                        agar        SP.  -           SP.  dark brown                                   (Ty)        Spg. -           Spg. -                                                             tyrosine crystals                                                                               tyrosine crystals                                             dissolved under  not dissolved                                                 mycelium                                                                  G.   good        G.   good        G.   good to very good           Oat-yeast   SM.  dark brown  Spg. ++, frost-like                                                                             SM.  orange                      agar                              on the SM.                                   (OY)        SP.  light brownish               SP.  -                                       Spg. +                            Spg. ++, frost-like                          G.   good to very good            G.   very good                   "Emerson"   SM.  brown to brown-black         SM.  orange                      agar        SP.  ochre with greenish tint     SP.  -                           (E)         Spg. +                            Spg. ++, frost-like              __________________________________________________________________________                 SE 100           SE 101           SK 2                             __________________________________________________________________________     Casamino-   G.   good        G.   good                                         peptone     SM.  pale ochre  SM.  red                                          Czapek      AM.  ++, white   AM.  few, white                                   agar (CPC)  SP.  -           SP.  -                                                        G.   good        G.   bulky                                        Czapek      SM.  colorless   SM.  colorless to pink                            agar        AM.  ++, white   AM.  +, white                                     (Cz)        SP.  -                                                                         G.   good        G.   good                                         "Emerson"   SM.  colorless to                                                                               SM.  pale red to red,                             agar             pale ochre       later paling                                 (E)         AM.  ++, white   AM.  -                                                        SP.  -           SP.  -                                                        G.   good        G.   good        G.   bulky                       Oat-yeast   SM.  pale ochre  SM.  pink        SM.  brown                       agar        AM.  ++, white   AM.  ++, dirty pink                               (OY)        SP.  -           SP.  yellowish-brownish                                                                         SP.  -                                                        Spg. ++                                                                                         G.   good (relatively)           Milk agar                                     SM.  golden yellow               (Ca                                                surface knobby              Ca/2)                                         SP.  -                                                                         Spg. -                                                                              Casein peptonized                                                         G.   good                        Tyrosine                                      SM.  brownish gold-yellow                                                           to                          agar                                               yellow-orange               (Ty)                                          SP.  golden brown to                                                                reddish                                                                        brown                                                                     Spg. -                                                                              tyrosine crystals                                                              dissolved                   __________________________________________________________________________

                                      Table 4                                      __________________________________________________________________________     Growth in Various Carbon Sources                                                        SB2 SB5 SB11                                                                               SB12                                                                               SB18                                                                               SB27                                                                               SB46                                                                               SE21                                                                               SE39                                                                               SE50                                                                               SE55                          __________________________________________________________________________     Glucose  ++  ++  ++  ++  ++  ++  ++  ++      ++  ++                            Fructose ++      ++  ++  ++  ++  ++  ++  ++  ++  ++                            Saccharose                                                                              ++  ++  ++  ++  ++  ++  ++  ++      ++  ++                            Mannitol ++  ++  ++  ++  ++  ++  ++  -       ++  ++                            L-Arabinase                                                                             ++  ++  ++  ++  ++  ++  ++  ++      ++  ++                            Raffinose                                                                               -   -   ++  -   -   -   -   -       +   +                             Inositol -   -   ++  -   ++  ++  ++  -       -   -                             D-Xylose ++  ++  ++  ++  ++  ++  ++  ++  ++  ++  ++                            __________________________________________________________________________      ++ Growth as good as on glucose or better                                       + Growth worse than on glucose, but better than on sugar-free mineral         salt-agar                                                                       - Growth as on mineral salt-agar without carbon source                  

The strains set forth in Table 1 were either drawn from established culture collections such as the American Type Culture Collection and the Centraalbureau voor Schimmelcultures, or deposited under the CBS-numbers set forth above in the Centraalbureau voor Schimmelcultures in Baarn, Holland.

Tables 2 and 3 list some characteristics of the strains described.

To obtain the glycoside-hydrolase inhibitors the strains listed above are cultured in the nutrient solutions described above. In doing so it should be noted that for optimum production practically every strain requires a different nutrient solution of different qualitative and quantitative composition.

After 1 to 10 day's incubation at 15°-60°C., preferably 24°-50°C., in a shaking flask or in fermenters of different size, the mycelium is separated from the culture solution and, depending on the occurrence of the inhibitors, the active principle is concentrated using the culture solution and/or the mycelium.

The inhibitors are obtained from the culture broths by lyophilization or precipitation with salts or water-soluble organic solvents (such as, for example, lower alcohols and ketones) or by adsorption of the active substances on ion exchangers.

The inhibitors are obtained from the mycelia by extraction with organic solvents, such as, for example, alcohols, ketones, ethers, esters and sulphoxides.

For this purpose, the fermentation batch is centrifuged at 3,000-20,000 revolutions per minute, preferably 6,000-10,000 revolutions per minute, for 10-60 minutes, preferably 30 minutes, or is filtered, preferably under pressure and with the help of filter aids, such as, for example, Claricel, and is thus separated into culture broth and mycelium residue.

The inhibitor can be isolated from the particular culture broth in various ways:

a. Concentration of the culture broths under reduced pressure (10-50 mm Hg) at bath temperatures of 20°-100°C., preferably 40°-80°C., to approximately 1/5 to 1/50 of the initial volume. The concentrated extract is filtered or centrifuged and the clear filtrate (or the clear supernatant liquid) is lyophilized, if required after prior desalination.

b. Precipitation of the inhibitors from the culture broth [or from the culture broths concentrated according to (a)] by adding water-soluble organic solvents, such as, for example, alcohols or ketones, preferably methanol, ethanol, or acetone, up to a content of 60-90 percent. Since inactive concomitant substances are precipitated at low concentration of solvents, this precipitation process is particularly suitable for fractional precipitation to remove undesired concomitant substances.

c. Salting-out of the inhibitors from the extracts [or from the extracts concentrated according to (a) ], for example, with ammonium sulphate, sodium chloride and the like. The precipitate formed is collected by centrifuging or filtering and is either directly washed with acetone and ether and dried in vacuo or redissolved in water, dialyzed and lyophilized.

d. Adsorption of the inhibitors on ion exchangers. This process is suitable for isolating those inhibitors which, because of their chemical nature, carry a charge. The inhibitor is desorbed by changing the ionic strength or the pH value of the elution medium.

In addition to the inhibitor, undesired concomitant substances are frequently present in the culture broths. These concomitant substances can be separated off in various ways, for example, by denaturing the concomitant substances by means of heat in the case of inhibitors which are heat-stable, or by dialysis through appropriate membranes in the case of low molecular inhibitors, in which case the undesired concomitant substances are retained by the membrane, or by fractional precipitation [compare (b)] or by adsorption of the concomitant substances on ion exchangers.

The inhibitors are obtained from the mycelia by repeated extraction of the mycelium with organic solvents, preferably two extractions of 10-20 minutes with 3-5 volumes of acetone (relative to the moist mycelium volume) and subsequent single extraction of 5-10 minutes with ether. The mycelium extracted in this way is dried in vacuo and subsequently extracted for 2-8 hours with 3-10 parts by weight of dimethyl sulphoxide, while stirring, and thereafter centrifuged at 10,000 to 20,000 revolutions per minute. The acetone extracts and ether extracts are concentrated to dryness in vacuo and taken up with the dimethyl sulfoxide (DMSO) extract.

Instead of extracting the dry mycelium powder with dimethyl sulfoxide (DMSO), it can also be extracted over a longer period, preferably 12-24 hours, with water or dilute electrolyte solutions.

The new substances dissolve well in water. One group of the inhibitors is heat-stable at neutral pH values, stable to acid (pH 2), stable to alkali (pH 12), and slowly dialyzable. These inhibitors are not inactivated by trypsin and pepsin and, in turn, do not inhibit the enzymes mentioned. They cannot be dyed with the typical protein dyes and do not show a characteristic absorption in the UV up to 250 nm. These inhibitors cannot be inactivated with urea and β-mercaptoethanol. According to estimates from gel filtration, the molecular weight of these inhibitors is above 500 but below 6,000. On hydrolytic splitting, monosaccharides, for example, glucose, are obtained. According to these findings, these inhibitors are oliogosaccharides or polysaccharides or their derivatives.

The best inhibitors of this group show inhibition activities, against amylase, of 8,000 AIU/mg.

Another group of inhibitors is heat-labile and not dialyzble, or hardly dialyzable. These inhibitors are inactivated more or less rapidly by trypsin. Urea and α-mercaptoethanol also inactivate most of these inhibitors. These inhibitors are probably substances of peptide character.

The best inhibitors of this group show inhibiting activities, against amylase, of 80 AIU/mg.

It is known that in the case of animals and man hyperglycemias arise after the intake of foodstuffs and beverages containing carbohydrates (for example, corn starch, potato starch, fruit, fruit juice or chocolate), these hyperglycemias resulting from a rapid degradation of the carbohydrates by glysoside-hydrolases (for example, salivary and pancreatic amylases, maltases and saccharases) in accordance with the following equation:

             amylase            maltase                                            starch   →  maltose  → glucose or                                or glycogen                                                                             saccharase                                                            sucrose  →  glucose + fructose                                     

These hyperglycaemias are of particularly strong and lasting character in the case of diabetics. In adipose cases, alimentary hyperglycemia frequently causes a particularly strong secretion of insulin, which, in turn, leads to increased synthesis of fat and reduced degradation of fat. In connection with such hyperglycemias, a hypoglycemia frequently occurs in metabolically healthy and adipose persons as a result of the insulin secretion. It is known that not only hypoglycemias but also chyne remaining in the stomach stimulate the production of gastric juice, which for its part participates or encourages the formation of a gastritis, or a gastric or duodenal ulcer.

It has now been found that inhibitors of glycoside-hydrolases, according to the invention, obtained and isolated in accordance with the above methods, considerably reduce alimentary hyperglycemia, hyperinsulinemia and hypoglycemia after dosing rats and/or man with wheat starch or sucrose or maltose, and speed up the passage of these carbohydrates through the stomach.

Furthermore, it is known that carbohydrates, especially sucrose, are split by microorganisms in the mouth cavity and that caries formation is promoted thereby.

Inhibitors of glycoside-hydrolases are, therefore, suitable for use as therapeutic agents for the following indications: obesity, adiposit, hyperlipoidemia (arteriosclerosis) diabetes, pre-diabetes, gastritis, gastric ulcer, duodenal ulcer, and caries.

The present invention, therefore, provides a pharmaceutical composition containing as active ingredient an inhibitor of the invention in admixture with a liquid diluent other than a solvent of a molecular weight less than 200 (preferably less than 350) except in the presence of a surface active agent.

The invention further provides a pharmaceutical composition containing as active ingredient an inhibitor of the invention in the form of a sterile or isotonic aqueous solution.

The invention also provides a medicament in dosage unit form comprising an inhibitor of the invention either alone or in a admixture with a diluent.

The invention also provides a medicament in the form of tablets, (including lozenges and granules), dragees, capsules, pills, ampoules and suppositories comprising an inhibitor of the invention either alone or in admixture with a diluent.

"Medicament" as used in this specification means physically discrete coherent portions suitable for medical administration. "Medicament in dosage unit form" as used in this specification, means physically discrete coherent portions suitable for medical administration each containing a unit dose or a multiple (up to four times) or submultiple [down to a fortieth (1/40)] of a unit dose of the inhibitor of the invention. Whether the medicament contains a unit dose or, for example, a half (1/2), a third (1/3), or a quarter (1/4) of a unit dose will depend on whether the medicament is to be administered once, for example, twice, three times or four times a day, respectively.

A unit dose is the amount of inhibitor to be taken on one occasion.

The pharmaceutical compositions according to the invention may, for example, take the form of gels, pastes (e.g., toothpastes), creams, chewing-gums, suspensions, solutions and emulsions of the active ingredient in aqueous or non-aqueous diluents, syrups, granules or powders.

The diluents to be used in pharmaceutical composition (e.g., granulates) adapted to be formed into tablets, dragees, capsules and pills include the following: (a) fillers and extenders, e.g., starch, sugars, mannitol, and silicic acid; (b) binding agents, e.g., carboxymethyl cellulose and other cellulose derivatives, alginates, gelatine, and polyvinyl pyrrolidone; (c) moisturizing agents, e.g., glycerol; (d) disintegrating agents, e.g., agar-agar, calcium carbonate, and sodium bicarbonate, (e) agents for retarding dissolution, e.g., paraffin; (f) resorption accelerators, e.g., quaternary ammonium compounds; (g) surface active agents, e.g., cetyl alcohol, glycerol monostearate; (h) adsorptive carriers, e.g., kaolin and bentonite; (i) lubricants, e.g., talc, calcium and magnesium stearate, and solid polyethylene glycols; (j) elastomeric binders such as chicle.

The tablets, dragees, capsules and pills formed from the pharmaceutical compositions of the invention can have the customary coatings, envelopes and protective matrices, which may contain opacifiers. They can be so constituted that they release the active ingredient only or perferably in a particular part of the intestinal tract, possibly over a period of time. The coatings, envelopes and protective matrices may be made, for example, of polymeric substances or waxes.

The ingredient can also be made up in microencapsulated form together with one or several of the above-mentioned diluents.

The diluents to be used in pharamceutical compositions adapted to be formed into suppositories can, for example, be the usual water-soluble or water-insoluble diluents, such as polyethylene glycols and fats [e.g., cocoa oil and high esters (e.g., C₁₄ -alcohol and C₁₆ -fatty acid)] or mixtures of these diluents.

The pharmaceutical compositions which are pastes, creams, and gels can, for example, contain the usual diluents, e.g., animal and vegetable fats, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide or mixtures of these substances.

The pharmaceutical compositions which are powders can, for example, contain the usual diluents, e.g., lactose, talc, silicic acid, aluminum hydroxide, calcium silicate, and polyamide power or mixtures of these substances.

The pharmacuetical compositions which are solutions and emulsions can, for example, contain the customary diluents (with, of course, the above-mentioned exclusion of solvents having a molecular weight below 200 except in the presence of a surface-active agent), such as solvents, dissolving agents and emulsifiers; specific examples of such diluents are water, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (for example, ground nut oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitol or mixtures thereof.

For parenteral administration, the solutions and emulsions should be sterile, and, if appropriate, blood-isotonic.

The pharmaceutical compositions which are suspensions can contain the usual diluents, such as liquid diluents, e.g., water, ethyl alcohol, propylene glycol, surface-active agents (e.g., ethoxylated isostearyl alcohols, polyoxyethylene sorbite and sorbitane esters), microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth or mixtures thereof.

All the pharmaceutical compositions according to the invention can also contain coloring agents and preservatives as well as perfumes and flavoring additions (e.g., peppermint oil and eucalyptus oil) and sweetening agents (e.g., saccharin). In particular, chewing gums and toothpastes will contain flavoring agents.

The pharmaceutical compositions according to the invention preferably contain about 0.1 to 99.5, more preferably from about 0.5 to 95 percent of the inhibitor by weight of the total composition.

In addition to an inhibitor of the invention, the pharmaceutical compositions according to the invention can also contain other pharmaceutically active compounds. They may also contain a plurality of different inhibitors of the invention. Particular examples of such other pharmaceutically active compounds are oral antidiabetic agents such as β-cytotropic sulphonyl-urea derivatives and biguanides which influence the blood sugar level.

The diluent in the medicament of the present invention may be any of these mentioned above in relation to the pharmaceutical compositions of the present invention. Such medicament may include solvents of molecular weight less than 200 as sole diluent.

The discrete coherent portions constituting the medicament according to the invention (whether in dosage unit form or net) may be, for example, any of the following: tablets (including lozenges and granules), pills, dragees, capsules, suppositories, and ampoules. Some of these forms may be made up for delayed release of the inhibitor. Some, such as capsules, include a protective envelope which renders the portions of the medicament physically discrete and coherent.

The preferred unit dose for the medicaments of the invention is 5,000-500,000 AIU, 2.5-250 MIU, or 100-10,000 SIU of inhibitor. A unit dose will be taken orally once or several times daily, usually immediately before, during, or after a meal.

It is envisaged that the inhibitor will be administered perorally. Preferred medicaments are, therefore, those adapted for peroral administration, such as tablets, dragees, and portions of chewing gum.

The toxicity of some of these inhibitors of glycosidehydrolases is extremely low. The active substance from Examples 14 and 37 was tolerated, without symptoms, at a dosage of 5 × 10⁶ AIU/kg of mouse or rat, administered orally. In the case of intravenous injection, mice and rats tolerated 10⁶ AIU/kg.

The following examples illustrate the production of the inhibitor according to the invention.

EXAMPLE 1

Three 1-liter Erlenmeyer flasks, containing 200 ml of a nutrient solution of composition: 2% starch, 1% glucose, 0.5% NZ-amines, 1.0% yeast extract and 0.4% CaCO₃ (sterilization: 30 minutes, 121°C.; pH adjusted to 7.2 before sterilization) are each inoculated with 1 ml of a primary culture (obtained in the same nutrient solution, inoculated from sloping test-tube cultures with oatmeal agar) of the strain SB 2, and the flasks are incubated at 28°C. on a rotary shaking machine. After a period of culture of 5.5 days, the contents of the three flasks are combined and the mycelium is separated off by centrifuging. 425 ml of supernatant liquor, containing 100 AIU/ml, are obtained.

The centrifuged supernatant liquor is concentrated to 60 ml on a rotary evaporator at 15-20 mm Hg and approximately 37°C. waterbath temperature. The viscous solution is stirred into 8 volumes = 480 ml of ethanol, while stirring. The precipitate formed is collected by centrifuging, again dissolved in 60 ml of water, and dialyzed for 6 hours against distilled water. The dialyzate is lyophilized. Yield: 1.6 g containing 19 × 10³ AIU/g.

EXAMPLE 2

Using a batch according to Example 1, 440 ml of centrifuged supernatant liquor containing 100 AIU/ml are obtained.

These 440 ml of centrifuged supernatant liquor are concentrated to 100 ml on a rotary evaporator. The concentrated solution is stirred into 8 volumes = 800 ml of ethanol and the precipitate is collected by centrifuging, twice washed with acetone and once with ether and dried in vacuo at room temperature. Yield: 2.2 g containing 15 × 10³ AIU/g.

EXAMPLE 3

Using a batch according to Example 1, 500 ml of a centrifuged supernatant liquor containing 120 AIU/ml are obtained after 5 days' culture. These 500 ml are lyophilized directly. Yield: 7.1 g containing 8.3 × 10³ AIU/g.

EXAMPLE 4

Three 1-liter Erlenmeyer flasks containing 200 ml of a nutrient solution of composition: 3% glycerine, 3% soya flour, and 0.2% CaCO₃ (sterilization: 30 minutes, 121°C.; pH after sterilization 7.2) are each inoculated with one ml of a primary culture (obtained in the same nutrient solution, inoculated from sloping test-tube cultures containing Czapek-peptone-casein agar) of the strain SB 12, and the flasks are incubated for 3 days at 28°C. on a rotary shaking machine. After the incubation, the contents of the flasks are combined and the mycelium is separated off by centrifuging. 500 ml of supernatant liquor containing 450 AIU/ml are obtained.

The supernatant liquor is treated with 250 g of ammonium sulphate added in portions while stirring and the mixture is subsequently centrifuged for 10 minutes at 12,000 revolutions per minute. The precipitate is dissolved in 100 ml of distilled H₂ O and 4 volumes (=400 ml) of acetone are added while stirring. A precipitate which sediments well is formed. The liquid is decanted off and the precipitate is washed twice with acetone and once with ether and dried in vacuo. Yield: 13.4 g containing 10 × 10³ AIU/g.

EXAMPLE 5

A precipitate obtained according to Example 4 after precipitation with ammonium sulphate is dissolved in 100 ml of water and dialyzed for 6 hours against distilled water. A dialyzate is obtained, which is frozen and lyophilized. Yield: 1.5 g containing 80 AIU/mg.

EXAMPLE 6

If a 1 liter Erlenmeyer flask is inoculated with 120 ml of a nutrient solution according to Example 1, from a sloping test-tube culture of the strain St. 19, a culture solution containing 25 AIU/ml is obtained after 3 days' culture at 28°C. on a rotary shaking machine.

EXAMPLE 7

If a mix according to Example 4 is incubated for 3 days at 32°C., a culture filtrate containing 350 AIU/ml is obtained after filtering off the mycelium.

EXAMPLE 8

If a nutrient solution according to Example 1 is inoculated in accordance with Example 4, culture filtrates containing 270 AIU/ml are obtained after 3 days' incubation at 28°C.

EXAMPLE 9

Six 1-liter Erlenmeyer flasks, each containing 100 ml of a nutrient solution consisting of 3% glucose, 3% soya flour, and 0.2% CaCO₃ (sterilization: 30 minutes, 121°C.; pH after sterilization 7.2) are each inoculated with 1 ml of primary culture (obtained as in Example 1) of the strain SB 5, and are incubated for four days at 28°C. on rotary shaking machines. The contents of the flasks are combined and the mycelium is separated off by centrifuging. The resulting 500 ml of supernatant liquor containing 150 AIU/ml are frozen and lyophilized. Yield: 6.9 g containing 11 × 10³ AIU/g.

EXAMPLE 10

If 24 flasks of a mix according to Example 4, each containing 120 ml, are inoculated with a primary culture of the strain SB 11 (obtained according to Example 4) and incubated for 5 days at 28°C., 2.0 liters of a supernatant liquor containing 1.1 MIU/ml are obtained after centrifuging. These 2 liters are concentrated to 200 ml on a rotary evaporator. The 200 ml of concentrate are dialyzed for 24 hours in a Visking dialysis tube (type 27/100 FT, Union Carbide Corporation) against 2 liters of distilled water, at room temperature. The outer medium, containing inhibitor, is concentrated to 100 ml on a rotary evaporator and is added dropwise to 900 ml of absolute ethanol, while stirring. The almost inactive precipitate which separates is centrifuged off and discarded, and the alcoholic supernatant liquor is concentrated to 30 ml on a rotary evaporator.

1 ml of this concentrate contains 70 MIU/ml.

For further purification, this solution is passed over an anion exchange column (Amberlite IRA 410, acetate form, in 0.05 M NH₄ -acetate, pH 7, 2.5 × 20 cm column) and the active fractions are combined and gel-filtered on Sephadex ^(R) G 75 in H₂ O. The active fractions from the gel filtration are concentrated to 12 ml. 1 ml of this solution contains 150 MIU/ml.

The mycelium (˜500 ml) was twice extracted with 1 liter of acetone and once with 1 liter of ether and the extracts were combined and evaporated to dryness on a rotary evaporator in vacuo. The mycelium residue was dried in vacuo at 20°C. and the resulting dry mycelium powder (˜51 g) was subsequently extracted for 2 hours at room temperature with 150 ml of dimethyl sulfoxide (DMSO). After centrifuging (30 minutes, 15,000 r.p.m.) the acetone/ether extract which has been concentrated to dryness is taken up with the dimethyl sulfoxide (DMSO) supernatant liquor from the dry mycelium powder. Yield: 120 ml containing 3 MIU/ml.

EXAMPLE 11

If a 1 liter Erlenmeyer flask containing 120 ml of a nutrient solution according to Example 1 is inoculated according to Example 9 and incubated for 6 days at 28°C. on a rotary shaking machine, a culture filtrate containing 0.9 MIU/ml is obtained.

EXAMPLE 12

If five 1-liter Erlenmeyer flasks, each containing 120 ml of a nutrient solution of composition: 2.5% starch, 0.5% glucose, 0.5% NZ-amines, 1.0% yeast extract and 0.4% CaCO₃ (sterilization: 30 minutes, 121°C.; pH adjusted to 7.2 before sterilization) are each inoculated with 2 ml of a primary culture (obtained according to Example 1) of the strain SB 27 and these flasks are incubated for 4 days at 28°C. on a rotary shaking machine, 500 ml of supernatant liquor containing 70 AIU/ml are obtained after combining the flasks and centrifuging off the mycelium. The centrifuged supernatant liquor is frozen and lyophilized. Yield: 7.7 g containing 3.5 × 10³ AIU/g.

EXAMPLE 13

If a mix according to Example 1 is inoculated with 2 ml of a primary culture of the strain SB 18 (obtained according to Example 1) and incubated for 3 days at 28°C., a culture broth containing 1100 AIU/ml, 0.17 MIU/ml and 1.0 SIU/ml is obtained.

EXAMPLE 14

If five experimental fermenters each containing 8 liters of culture solution according to Example 1 are each inoculated with 120 ml of a primary culture (obtained according to Example 1) and incubated for 65 hours at 28°C. while stirring and aerating, 30 liters of culture broth are obtained after combining the fermentation broths and separating off the mycelium. These 30 liters of centrifuged culture broth (0.57 MIU/ml, 6.2 SIU/ml, and 8,000 AIU/ml) are concentrated to 5 liters in vacuo at 20 mm Hg and 100°C. bath temperature, 4 volumes (=20 liters) of acetone are added to the concentrate while stirring, and the black smeary precipitate which forms is collected by centrifuging at 6,000 r.p.m. for 30 minutes; the precipitate is dissolved in 2.5 liters of water and the black-colored solution is stirred for 60 minutes with 500 g of moist Amberlite [IRA 410 (acetate form, pH 7)]. The mixture is separated into supernatant liquor and Amberlite sediment by centrifuging for 10 minutes at 6,000 r.p.m. In the same way, the supernatant liquor is further stirred three times, in each case with 500 g of Amberlite for 60 minutes, and subsequently with a further 500 g of Amberlite overnight (˜15 hours). After this treatement, the supernatant liquor shows a light yellow coloration, while the black concomitant dyestuffs were bonded to the ion exchanger. The collected Amberlite residues are twice washed with 1.5 liters of water and these wash waters are combined with the supernatant liquor containing inhibitor. The supernatant liquor, combined with the wash water, is concentrated to 1 liter in a rotary evaporator at 15 mm Hg and 80°C. bath temperature and subsequently added dropwise to 10 liters of acetone, with vigorous stirring. Hereupon, a white flocculent precipitate results, which is filtered off, washed with acetone and ether and dried in vacuo. Yield: 150 g of a white powder containing 1 × 10⁶ AIU/g and 450 SIU/g.

EXAMPLE 15

If, in a nutrient solution according to Example 1, the glucose is replaced by other sugars or sugar alcohols and shaking flasks each containing 120 ml of culture solution are each inoculated with 1 ml of a primary culture of the strain SB 18 (manufactured according to Example 1), culture solutions containing the following amylase inhibitor concentrations are obtained after 3 or 4 days' culture at 28°C. on rotary shaking machines:

    Additive    AIU/ml        AIU/ml                                               1% Each     after 3 days  after 4 days                                         ______________________________________                                         sucrose     5,400         10,500                                               lactose     9,100          4,600                                               maltose     7,500         13,600                                               galactose   9,100         10,200                                               glucose     7,000          9,700                                               sorbitol    2,600         10,200                                               mannitol    8,500          8,700                                               inositol    4,800          5,600                                               starch      6,400         10,200                                               ______________________________________                                    

EXAMPLE 16

If a nutrient solution consisting of 3% soya flour, 2% starch, 1% glucose, and 0.2% CaCO₃ is inoculated and incubated in accordance with Example 15, a culture broth containing 5,600 AIU/ml is obtained after four days' fermentation.

EXAMPLE 17

If a mix according to Example 13 is inoculated and incubated with a morphological variant of SB 18, the strain SB 18/5, a culture broth containing 27,400 AIU/ml is obtained after four days' fermentation.

The strain SB 18/5 was deposited at the Central Bureau voor Schimmelcultures in Baarn, Holland, under CBS No. 613.71.

EXAMPLE 18

If a mix according to Example 13 is inoculated and incubated with a morphological variant of SB 18, the strain SB 18/4, a culture broth containing 13,900 AIU/ml is obtained after four days' fermentation.

The strain SB 18/4 was deposited at the Central Bureau voor Schmmelcultures in Baarn, Holland, under CBS no. 612.71.

EXAMPLE 19

If a nutrient solution of composition: 2% starch, 1% glucose, 0.3% glycine, 0.25% corn-steep liquor, 0.4% soya flour, 0.1% NaCl, 0.1% K₂ HPO₄, 0.01% FeSO₄, and 0.01% CaCO₃ is inoculated and incubated in accordance with Example 12, a culture broth containing 2,900 AIU/ml is obtained after three days' incubation.

EXAMPLE 20

If 2 flasks of a mix according to Example 1 are inoculated with 1 ml of a primary culture of the strain SB 46 and incubated for 4 days at 28°C. on a rotary shaking machine, 250 ml of supernatant liquor containing 250 AIU/ml are obtained after centrifuging.

These 250 ml of supernatant liquor are treated with 150 g of ammonium-sulphate added in portions while stirring and the mixture is subsequently centrifuged for 15 minutes at 10,000 r.p.m. The residue is dissolved in 12 ml of H₂ O and dialyzed for three hours against distilled water. The 20 ml of dialysate are precipitated with 6 volumes (= 120 ml) of acetone in an ice bath and the precipitate is filtered off, washed with acetone and ether and subsequently dried in vacuo. Yield: 0.28 g containing 220 × 10³ AIU/g.

EXAMPLE 21

If a 1 liter Erlenmeyer flask containing 120 ml of a nutrient solution according to Example 1 is inoculated with 2 ml of a primary culture of the strain SE 5 (produced according to Example 4) and incubated for 7 days on a rotary shaking machine at 28°C., a culture broth containing 0.09 MIU/ml is obtained.

The mycelium is treated with 50 ml of acetone, homogenized for one minute on an Ultraturrax homogenizer (Messrs. Janke and Kunkel, Staufen, Breisgau) and the mixture is subsequently centrifuged for 10 minutes at 3,000 r.p.m. The residue is again extracted, in the same way, with 50 ml of acetone and subsequently extracted once with 50 ml of ether, and the three extracts are combined and concentrated almost to dryness in a rotary evaporator at approximately 10-20 mm Hg and a waterbath temperature of 37°C. The mycelium residue is dried in vacuo and subsequently treated with 15 ml of dimethyl sulphoxide (DMSO), homogenized for two minutes by means of the Ultraturrax and extracted for two hours while stirring (magnetic stirrer). Thereafter the mixture is centrifuged for 30 minutes at 20,000 r.p.m. The dimethyl sulfoxide (DMSO) extract is then decanted from the extracted mycelium, added to the residue of the acetone/ether extraction, and the mixture stirred for approximately 30 minutes (magnetic stirrer), and again centrifuged for 10 minutes at 20,000 r.p.m., and the clear supernatant liquor is tested as mycelium extract. 0.14 MIU/ml.

EXAMPLE 22

If two shaking flasks according to Example 1, each containing a charge of 160 ml, are each inoculated and incubated with 1 ml of a primary culture (according to Example 1) of the strain SE 21, 200 ml of a supernatant liquor containing 360 AIU/ml are obtained after centrifuging.

The centrifuged supernatant liquor is treated with 100 g of ammonium-sulphate added in portions while stirring and is centrifuged, the precipitate is dissolved in 20 ml of water and the solution is precipitated with 2 volumes (ca 40 ml) of acetone. The precipitate is washed with acetone and ether and dried in vacuo. Yield: 2 g containing 25 × 10³ AIU/g.

Example 23

If a mix according to Example 14 is inoculated and incubated with primary cultures of the strain SE 21, culture broths containing 140 AIU/ml are obtained after 65 hours' fermentation.

Example 24

If a 1 liter Erlenmeyer flask containing 120 ml of a nutrient solution of the composition according to Example 4 is inoculated with 1 ml of a primary culture, according to Example 1, of the strain SE 39 and incubated in accordance with Example 1, a culture solution containing 50 AIU/ml is obtained after only 3 days' fermentation.

EXAMPLE 25

If glycerine is replaced by glucose in a mix according to Example 24, culture solutions containing 60 AIU/ml are obtained after 3 days.

EXAMPLE 26

If in a mix according to Example 24 the nutrient solution according to Example 1 is modified and the starch replaced by glucose, culture solutions containing 140 AIU/ml are obtained after three days' incubation.

EXAMPLE 27

If five 1-liter Erlenmeyer flasks each containing 120 ml of nutrient solution of composition: 3% glucose, 0.5% NZ-amines, 1.0% yeast extract and 0.4% CaCO₃ (sterilization: 30 minutes, 121°C.; pH before sterilization 7.2) are inoculated with 1 ml of a primary culture of the strain SE 39 (manufactured according to Example 4) and incubated for three days on a rotary shaking machine at 28°C., 500 ml of a supernatant liquor containing 160 AIU/ml are obtained after centrifuging.

The 500 ml of the supernatant liquor are precipitated with 6 volumes (= 3 liters) of acetone while stirring and the light brown precipitate is filtered off, washed with acetone and ether and dried in vacuo. Yield: 3.5 g containing 13.5 × 10³ AIU/g.

EXAMPLE 28

If an Erlenmeyer flask containing 120 ml of a nutrient solution according to Example 4 is inoculated with 1 ml of a primary culture of the strain SE 50 (primary culture manufactured according to Example 4) and incubated, again in accordance with Example 4, a culture solution containing 1.0 SIU/ml is obtained after 5 days.

EXAMPLE 29

If Example 28 is followed but with a nutrient solution according to Example 1, a culture solution containing 26,000 AIU/ml and 0.18 MIU/ml and 1.8 SIU/ml is obtained after 5 days' incubation.

EXAMPLE 30

If Example 29 is followed but with a flask charge of 200 ml, a culture solution containing 24,500 AIU/ml and 0.18 MIU/ml and 2.1 SIU/ml is obtained after 5 days' incubation.

Example 31

If Example 28 is followed, but with a nutrient solution of composition: 2% starch, 1% glucose, 0.3% glycine, 0.25% cornsteep liquor, 0.4% soya flour, 0.1% NaCl, 0.1% K₂ HPO₄, 0.01% MgSO₄, 0.01% CaCO₃ and 0.01% FeSO₄, a culture solution containing 12,900 AIU/ml is obtained after five days' fermentation.

EXAMPLE 32

If, in a nutrient solution according to Example 1, the glucose is replaced by other sugars of sugar alcohols and the solution inoculated and incubated in accordance with Example 28, culture solutions containing the following amylase inhibitor concentrations are obtained:

    Additives   AIU/ml        AIu/ml                                               (1%)        after 3 days  after 5 days                                         ______________________________________                                         sucrose     --            23,000                                               lactose     24,000        --                                                   maltose     23,500        --                                                   galactose   31,300        --                                                   glucose     41,000        --                                                   sorbitol    24,000        --                                                   mannitol    33,000        --                                                   inositol    21,000        --                                                   starch      33,900        --                                                   ______________________________________                                    

EXAMPLE 33

If, in a mix according to Example 28, the glycerine is replaced by glucose, culture solutions containing 330 AIU/ml are obtained.

EXAMPLE 34

If, in a mix according to Example 28, the starch is replaced by glucose, culture solutions containing 660 AIU/ml are obtained.

EXAMPLE 35

If, in mixes according to Example 29, NZ-amines are replaced by other complex sourcss of nitrogen, culture solutions containing the following amylase inhibitor units are obtained after three days' fermentation:

           Substitute AIU/ml                                                       ______________________________________                                                soya flour 13,500                                                              fish solubles                                                                             16,800                                                              tryptone   25,200                                                              meat extract                                                                              32,700                                                              pharmamedia                                                                               23,100                                                       ______________________________________                                    

EXAMPLE 36

If a mix according to Example 29 is inoculated with a morphological variant of the strain SE 50, namely strain SE 50/12, culture broths containing 51,500 AIU/ml are obtained after three days' fermentation.

EXAMPLE 37

If five experimental fermenters each containing 8 liters oc culture solution according to Example 1, but with 0.1 percent by volume of Bayer E 100 anti-foaming agent, are each inoculated with 120 ml of a primary culture (obtained according to Example 1) of strain SE 50 and incubated for 65 hours at 28°C. while stirring and aerating, the fermentation mixes are combined and the mycelium is separated off, 24 liters of culture broth containing 13,000 AIU/ml and 6 SIU/ml are obtained. The culture broth is concentrated to 1 liter by evaporating in vacuo at 20 mm Hg and 100°C. bath temperature. The black-colored concentrate was centrifuged for 60 minutes at 15,000 r.p.m., the dark supernatant liquor was applied to an Amberlite IRA 410 column (9 cm φ, 50 cm height, Amberlite IRA 410 acetate, pH 7 in H₂ O), the column was eluted with 50 ml of H₂ O/hour and 20 ml of fractions were collected in the fraction collector. The light yellow-colored fractions containing inhibitor (a total of 300 = 6 liters) were combined, concentrated to a 1 liter on a rotary evaporator and added dropwise, with vigorous stirring, to 10 liters of absolute alcohol, whereupon a flocculent, almost white precipitate separates out. The precipitate is filtered off, washed with absolute alcohol and thereafter with ether and dried in vacuo. Yield: 60 g of a white powder containing 3 × 10⁶ AIU/g and 400 SIU/g.

EXAMPLE 38

The bulk of the inactive concomitant substances, especially the dark dyestuff, can also be separated off by precipitation with an equal volume of methanol. For this purpose, 26 liters of the centrifuged culture broth (produced according to Example 37, but with 0.02 percent by volume of Bayer Silicone E antifoaming agent) (43,000 AIU/ml) are concentrated to 1.2 liters by evaporation in vacuo at 20 mm Hg and 100°C. bath temperature. The deep black solution is treated with an equal volume (1.2 liters) of methanol while stirring, whereupon a flocculent black precipitate forms. It is filtered through a fluted filter and the brown filtrate (2.2 liters) is added dropwise to 10 liters of the dry spirit, while stirring vigorously, A light brown precipitate forms, which is filtered off, washed with acetone and ether and dried in vacuo. Yield: 185 g of a light ochre-colored powder containing 3,700 AIU/mg.

For further purification, the entire precipitate (185 g) is dissolved in 250 ml of water and the dark brown solution is passed over a 5 × 75 cm column of Amberlite IRA 410 (acetate form, pH 7). The column is eluted with water, the first 500 ml of eluate are discarded as first runnings and the next 5,000 ml of almost colorless eluate contain the inhibitor activity. They are concentrated to 200 ml on a rotary evaporator and the inhibitor is obtained therefrom by adding the solution dropwise to 2 liters of absolute ethyl alcohol, filtering off the precipitate, washing with ethanol and ether and drying in vacuo. Yield: 80 g of a white powder containing 8,000 AIU/mg.

EXAMPLE 39

If a 1 liter Erlenmeyer flask containing 120 ml of a nutrient solution according to Example 4 is inoculated with 2 ml of a primary culture of the strain SE 55 (manufactured according to Example 4) and the culture is incubated for 6 days on a rotary shaking machine at 28°C., a culture broth containing 30 AIU/ml and 0.15 MIU/ml and 0.5 SIU/ml is obtained after separating off the mycelium.

If the mycelium is extracted in accordance with Example 20, an extract containing 15 AIU/ml, 0.17 MIU/ml and 0.5 SIU/ml is obtained.

EXAMPLE 40

If Example 39 is followed, but using a nutrient solution according to Example 1, a culture solution containing 60 AIU/ml, 0.16 MIU/ml and 0.65 SIU/ml and a mycelium extract containing 30 AIU/ml and 0.16 MIU/ml and 0.33 SIU/ml are obtained.

EXAMPLE 41

If a nutrient solution according to Example 39 is inoculated from a sloping test-tube culture on oatmeal agar of the strain SS 26, and the culture is incubated in accordance with Example 40 for 5 days, an extract containing 0.14 MIU/ml is obtained after separating off the culture solution after extraction of the mycelium according to Example 21.

EXAMPLE 42

If Example 41 is followed using a culture of the strain SS 45, a mycelium extract containing 0.1 MIU/ml is obtained.

EXAMPLE 43

If Example 41 is followed using a culture of the strain SS 53, a culture broth containing 0.05 MIU/ml and a mycelium extract containing 0.07 MIU/ml is obtained.

EXAMPLE 44

If a 1 liter Erlenmeyer flask is inoculated with 120 ml of a nutrient solution of composition: 0.5% Bactopeptone, 0.5% meat extract, 0.2% yeast extract, 0.03% casein hydrolysate, 1% i-inositol, 1% sorbitol, 1% D-mannitol, 1% glucose, 0.1% K₂ HPO₄, 0.05% MgSO₄, 0.05% KCl and 0.01% FeSO₄, pH adjusted to 7.2, is inoculated from a sloping agar culture (manufactured according to Example 1) of the strain SS 51, a culture solution containing 0.072 MIU/ml and a mycelium extract (manufactured according to Example 21) containing 0.054 MIU/ml are obtained after culture on a rotary shaking machine at 28°C. for 7 days.

EXAMPLE 45

If a nutrient solution according to Example 4 is inoculated from a sloping test-tube culture on oatmeal agar of the strain St 19 and is incubated for 3 days in accordance with Example 4, a culture solution containing 20 AIU/ml is obtained after separating off the mycelium.

EXAMPLE 46 Experimental technique for demonstrating the action of glycosidehydrolase inhibitors in rats and man

To produce alimentary hyperglycemia and hyperinsulinemia after feeding with carbohydrate, rats (n = 6) are given 2.5 g of starch or maltose or sucrose as a solution or suspension/kg, administered orally (control animals). Batches of 6 other rats receive, additionally to one of the above-mentioned carbohydrates, a glycoside-hydrolase inhibitor in the dosage indicated, administered orally. The blood sugar in the blood from the retro-orbital venous plexus is determined at short intervals after the administration of the carbohydrate by means of the Auto-Analyser [technicon, according to Hoffmann: J. biol. Chem. 120, 51 (1937)].

To produce an alimentary hyperglycemia in man, 50 g of starch/Vp. are administered orally as an aqueous suspension. The blood sugar is determined immediately before the start of the experiment, and at short intervals thereafter, in the capillary blood from the fingertip, in the way described above. In a further experiment, the active substance is added to the starch suspension.

The insulin is determined at short intervals after the administration of the carbohydrate, in the serum of batches of 6 rats of which one group is given 2.5 g of starch/kg, administered orally (control) and the other groups are additionally given a glycoside-hydrolase inhibitor.

The determination of insulin in the serum is carried out by radio-immunology, based on the double antibody method of Hales and Randle [Biochem. J. 88, 137 (1963) ].

Determination of starch in the gestrointestinal tract of rats was carried out at short intervals after oral administration of 300 mg starch/rat. To this purpose, the individual sections of the digestive tract were, after sacrifice of the animal, prepared and washed, and the undigested starch contained in them determined as glucose after acid hydrolysis.

TABLE 1 (EXAMPLE 46)

Blood glucose in mg% (mean value ± 1s) of fasting rats at various times after oral administration of starch ± active substance from Example 5.

    ______________________________________                                                        15       60 mins.                                               ______________________________________                                         control without starch                                                                          61 ± 16  70 ± 3.4                                       control with starch                                                                            140 ± 11 145 ± 15                                        starch + 25,000 AIU/kg                                                                         106 ± 20 128 ± 11                                        ______________________________________                                    

TABLE 2 (EXAMPLE 46)

Blood glucose in mg% (mean value ± 1s) of fasting rats at various times after oral administration of starch ± active substance from Example 14.

    ______________________________________                                                       15      30        60 mins.                                       ______________________________________                                         control without starch                                                                         72 ± 8.5                                                                              78 ± 12                                                                               69 ± 12                                 control with starch                                                                           159 ± 28                                                                              153 ± 36                                                                              164 ± 7.9                                starch + 25,000 AIU/mg                                                                         95 ± 3.7                                                                              95 ± 8.7                                                                              96 ± 7.2                                               ========            ========                                    ______________________________________                                    

TABLE 3 (EXAMPLE 46)

Blood glucose in mg% (mean value ± 1s) of fasting rats at various times after oral administration of starch ± active substance from Example 37.

    ______________________________________                                                       15      30       60 mins.                                        ______________________________________                                         control without starch                                                                         72 ± 8.5                                                                              78 ± 12                                                                               69 ± 12                                 control with starch                                                                           159 ± 28                                                                              153 ± 36                                                                              164 ± 7.9                                starch + 25,000 AIU/kg                                                                         83 ± 6.5                                                                             105 ± 5.2                                                                             104 ± 6.5                                               ========            ========                                    ______________________________________                                                                         against control                                       P < 0.01 ========P < 0.001                                                                              with carbohydrate                         

TABLE 4 (EXAMPLE 46)

Blood glucose in mg% of a healthy test person at various times after oral administration of starch ± active substance from Example 37.

    ______________________________________                                                        0    15     30     60   90 mins.                                ______________________________________                                         starch without active substance                                                                 106    128    150  146  136                                   starch + 60,000 AIU/Vp                                                                          100    112    108  110  104                                   ______________________________________                                    

TABLE 5 (EXAMPLE 46)

Blood glucose in mg% (mean value ± 1s) of fasting rats at various times after oral administration of maltose ± active substance from Example 10.

    ______________________________________                                                       15      30       60 mins.                                        ______________________________________                                         control without maltose                                                                        70 ± 6.7                                                                              81 ± 15                                                                               72 ± 9.6                                control with maltose                                                                          160 ± 13                                                                              153 ± 11                                                                              172 ± 13                                 maltose + 1.5 MIU                                                                             120 ± 13                                                                              124 ± 12                                                                              134 ± 9.4                                               ========  ========  ========                                    ______________________________________                                    

TABLE 6 (EXAMPLE 46)

Blood glucose in mg% (mean value ± 1s) of fasting rats at various times after oral administration of sucrose ± active substance from Example 14.

    ______________________________________                                                       15      30       60 mins.                                        ______________________________________                                         control without sucrose                                                                        61 ± 16                                                                               77 ± 4.7                                                                              70 ± 3.4                                control with sucrose                                                                          125 ± 14                                                                              129 ± 7.9                                                                             135 ± 8.5                                sucrose + 50 SIU/kg                                                                           104 ± 4.1                                                                             116 ± 8.4                                                                             113 ± 3                                                           ----------                                                                               ========                                    ______________________________________                                                                        against control                                 ----P< 0.05                                                                               P< 0.01 ==P< 0.001  with carbohydrate                          

TABLE 7 EXAMPLE 46)

Serum insulin in μU/ml (means value ± 1s) of fasting rats at various times after oral administration of 2.5 g of starch ± active substance from Example 14; n = 6.

The serum insulin of fasting rats without being given starch was 6 ±1μU/ml in this experiment.

    ______________________________________                                         Mins. after admini-          Starch +                                          stration of starch                                                                            Starch control                                                                               10,000 AIU/kg                                     ______________________________________                                          5             52 ± 29    17 ± 8                                                                      --------                                          10             72 ± 20    15 ± 4                                                                      ====                                              20             44 ± 28    16 ± 7                                                                      --------                                          30             25 ±  8    17 ± 3                                         45             30 ±  5    13 ± 6                                                                      ====                                              60             21 ±  8    13 ± 8                                         120             9 ±  3    10 ± 3                                         180             8 ±  2     9 ± 2                                         ______________________________________                                    

TABLE 8 (EXAMPLE 46)

Serum insulin in μU/ml (mean value ± 1s) of fasting rats at various times after oral administration of 2.5 g of starch ± active substance from Example 37; n =6.

The serum insulin of fasting rats without being given starch was 6 ± 1μU/ml in this experiment.

    ______________________________________                                         Mins. after admini-          Starch +                                          stration of starch                                                                            Starch control                                                                               10,000 AIU/kg                                     ______________________________________                                          5             52 ± 29    18 ±  8                                        10             72 ± 20    24 ± 11                                                                     ====                                              20             44 ± 28    11 ±  5                                                                     --------                                          30             25 ±  8    13 ±  3                                        45             30 ±  5    18 ±  7                                        60             21 ±  8    15 ±  5                                        120             9 ±  3    13 ±  5                                        ______________________________________                                         180             8 ±  2    11 ±  5                                        ----P<0.05 P<0.01  ==P<0.001   against control with                                                           carbohydrate                               

FIGS. 1 TO 3 (EXAMPLE 46)

FIGS. 1 to 3 show the average content of starch in mg (ordinate)/rat (n = 6) in the stomach (FIG. 1), small intestine (FIG. 2) and large intestine (FIG. 3) at different times (abscissa), after oral application of 300 mg raw wheat starch/ rat by a throat probe. In FIGS. 1 to 3 curves 1 demonstrate the result for the controls (Group 1), which were given starch without amylase inhibitor, while the curves 2 demonstrate the result for animals which received an equal dose of starch with addition of 1000 AIU amylase inhibitor (Group 2).

RESULT:

The content of wheat starch is significantly smaller in the stomach (FIG. 1) 15 + 60 minutes after application (P< 0.001 to P< 0.02) in Group 2 with amylase inhibitor than in Group 1. The quicker passage of starch through the stomach after addition of amylase inhibitor can also be seen from FIG. 2. In this case the content of starch in the small intestine of Group 2 is significantly (P< 0.001) raised after 150-180 minutes. FIG. 3 shows that starch reaches the large intestine of rats undigested, if amylase inhibitor is added to the starch in the stated dose.

FIGS. 4 to 6 (Example 46)

In FIGS. 4 to 6 are shown the alteration of blood glucose in mg/100 ml (ordinate) (FIG. 4), the concentration of immunologically reacting insulin in μU/ml serum (ordinate) (FIG. 5), and the concentration of unesterified fatty acids in micro equivalents/liter of plasma (ordinate) (FIG. 6), against the initial value (0) as dependent on time (abscissa), after oral application of 60 g cooked starch for a human experimental subject. Curve 1 shows the course of the stated parameters (blood sugar, serum insulin, plasma UFA) in the control experiment. Curve 2 shows the change of these parameters after administration of the same dose of starch with the addition of 0.25 mg. AIU amylase inhibitor, curve 3 the changes in the stated parameters after addition of 0.5 mega AIU amylase inhibitor to 60 g cooked starch per person.

Results:

After temporary hyperglycemia between 0 + 30 minutes in FIG. 4 the blood glucose falls after 45-180 minutes to significantly under the initial value. As a result of the initial hyperglycemia the concentration of insulin in the serum in the control experiment (curve 1) climbs steeply. The addition of 0.25 (curve 2) or 0.5 mega AIU (curve 3) to the starch has the effect that the hypoglycemia (FIG. 4) as well as the hyperinsulemia (FIG. 5) is quickly weakened. The ratio of unesterified fatty acids in the plasma is substantially equal in all three experiments.

Example 47

Erlenmeyer flasks containing the medias given in the Table, are inoculated with the corresponding strains and shaken at the stated temperature on a rotary shaker. Thus are obtained, after several days, culture solutions, and, after working up according to Example 21, mycelium extracts, which have the activities given in the Table.

                                      Table                                        __________________________________________________________________________     (Example 47 )                                                                  Strain Number      Culture Solutions and Conditions of Growth                  Laboratory                                                                           Collection                                                                             Strain Name                                                                              See  Quantity                                                                            in Erlenmeyer                                                                          Growth Culture                       Reference                                                                              No.             Example                                                                             ml   Flask   Temperature                                                                           Period                        __________________________________________________________________________                                                      (Days)                        ST 50 CBS 693.69                                                                             Streptomyces                                                                             4   30   200    28         4                                         heimii                                                           ST 67 CBS 432.59                                                                             Streptomyces                                                                             44  30   200    28         4                                         tendae                                                           ST 45 CBS 434.51                                                                             Streptomoyces                                                                            4   30   200    28         4                                         aureofaciens                                                     RT 36 CBS 228.65                                                                             Chainia rubra                                                                            44  30   200    28         4                           RT 33 CBS 295.66                                                                             Chainia poonensis                                                                        44  30   200    28         5                           ST 12 NRRL B-2286                                                                            Streptomyces                                                                             1   30   200    28         3                                         murinus                                                          ST 51 CBS 498.68                                                                             Streptomyces                                                                             44  30   200    28         4                                         fradiae                                                          ST 3  NRRL 2580                                                                              Streptomyces                                                                             4   30   200    28         5                                         chrysomallus                                                     ST 1  ATCC 11523                                                                             Streptomyces                                                                             4   30   200    28         5                                         chrysomallus                                                     SS 55 CBS 624.71                                                                             Streptosporan-                                                                           1   120  1000   28         6                                         gium roseum                                                      SS 59 CBS 623.71                                                                             Streptosporan-                                                                           1   120  1000   28         6                                         gium amethy-                                                                   stogenes                                                         SS 62 CBS 625.71                                                                             Streptosporan-                                                                           1   120  1000   28         6                                         gium roseum                                                      AT 8  KCC-A 0027                                                                             Streptosporan4                                                                           120 1000 28     4                                                    gium viri-                                                                     dalbum                                                           AT 11 KCC-A 0025                                                                             Streptosporan-                                                                           4   120  1000   28         4                                 gium album                                                               AT 13 CBS 190.64                                                                             Ampullariella                                                                            4   120  1000   28         6                                         campanulata                                                      AT 14 CBS 193.64                                                                             Ampullariella                                                                            4   120  1000   28         4                                         regularis                                                        SE 89 CBS 619.71                                                                             Ampullariella                                                                            1   120  1000   28         6                                         spec.                                                            SE 100                                                                               CBS 622.71                                                                             Planomonospora                                                                           1   120  1000   28         6                                         spec.                                                            __________________________________________________________________________     Strain Number      Activity of Culture Filtrates U/ml                          Laboratory                                                                           Collection                                                                             Strain Name                                                                              Amylase Inhibition                                                                        Maltase Inhibition                                                                        Saccharase Inhibition            Reference                                                                              No.                                                                    __________________________________________________________________________     ST 50 CBS 693.69                                                                             Streptomyces                    0.06                                           heimii                                                           ST 67 CBS 432.59                                                                             Streptomyces         0.020      0.25                                           tendae                                                           ST 45 CBS 434.51                                                                             Streptomyces         0.002                                                     aureofaciens                                                     RT 36 CBS 228.65                                                                             Chainia rubra        0.005                                       RT 33 CBS 295.66                                                                             Chainia poonensis    0.003                                       ST 12 NRRL B-2286                                                                            Streptomyces                                                                             500        0.008                                                     murinus                                                          ST 51 CBS 498.68                                                                              Streptomyces                                                                            70                                                                   fradiae                                                          ST 3  NRRL 2580                                                                              Streptomyces                                                                             20                                                                   chrysomallus                                                     ST 1  ATCC 11523                                                                             Streptomyces                                                                             20                                                                   chrysomallus                                                     SS 55 CBS 624.71                                                                             Streptosporan-                                                                 gium roseum                                                      SS 59 CBS 623.71                                                                             Streptosporan-                                                                 gium amethy-                                                                   stogenes                                                         SS 62 CBS 625.71                                                                             Streptosporan-                                                                 gium roseum                                                      AT 8  KCC-A 0027                                                                             Streptosporan-                                                                 gium viri-                                                                     dalbum    dalbum                                                 AT 11 KCC-A 0025                                                                             Streptosporan-                                                                 gium album                                                       AT 13 CBS 190.64                                                                             Ampullariella                                                                            2000                                                                 campanulata                                                      AT 14 CBS 193.64                                                                             Ampullariella                                                                            500                                                                  regularis                                                        SE 89 CBS 619.71                                                                              Ampullariella                                                                 spec.                                                            SE 100                                                                               CBS 622.71                                                                             Planomonospora                                                                           50                                                                   spec.                                                            __________________________________________________________________________     Strain Number      Activity of the Mycelium Extract According to Examle                           21 (U/ml)                                                   Laboratory                                                                           Collection                                                                             Strain Name                                                                              Amylase Inhibition                                                                        Maltase Inhibition                                                                        Saccharase Inhibition            Reference                                                                              No.                                                                    __________________________________________________________________________     ST 50 CBS 693.69                                                                             Streptomyces                                                                   heimii                                                           ST 67 CBS 432.59                                                                             Streptomyces                                                                   tendae                                                           ST 45 CBS 434.51                                                                             Streptomyces                                                                   aureofaciens                                                     RT 36 CBS 228.65                                                                             Chainia rubra                                                    RT 33 CBS 295.66                                                                             Chainia poonensis                                                ST 12 NRRL B-2286                                                                            Streptomyces                                                                   murinus                                                          ST 51 CBS 498.68                                                                              Streptomyces                                                                  fradiae                                                          ST 3  NRRL 2580                                                                              Streptomyces                                                                   chrysomallus                                                     ST 1  ATCC 11523                                                                             Streptomyces                                                                   chrysoamallus                                                    SS 55 CBS 624.71                                                                             Streptosporan-                                                                           100                                                                  gium roseum                                                      SS 59 CBS 623.71 Streptosporan-                                                              70                                                                             gium amethy-                                                                   stogenes                                                         SS 62 CBS 625.71                                                                             Streptosporan-                                                                           150                                                                  gium roseum                                                      AT 8  KCC-A 0027                                                                             Streptosporan-                                                                           150                                                                  gium viri-                                                                     dalbum                                                           AT 11 KCC-A 0025                                                                             Streptosporan-                                                                           100                                                                  gium album                                                       AT 13 CBS 190.64                                                                             Ampullariella                                                                  campanulata                                                      AT 14 CBS 193.64                                                                             Ampullariella                                                                  regularis                                                        SE 89 CBS 619.71                                                                             Ampullariella                                                                            2000                                                                 spec.                                                            SE 100                                                                               CBS 622.71                                                                             Planomonospora                                                                           50                                                                   spec.                                                            __________________________________________________________________________     Strain Number      Culture Solutions and Conditions of Growth                  Laboratory                                                                           Collection                                                                             Strain Name                                                                              See  Quantity                                                                            In Erlenmeyer                                                                          Growth Culture                       Reference                                                                              No.             Example                                                                             ml   Flask   Temperature                                                                           Period                        __________________________________________________________________________                                                      (Days)                        AT 4  CBS 191.64                                                                             Ampullariella                                                                            4    120  1000    28     6                                           digitata                                                         AT 9  KCC-A 0028                                                                             Streptosporan-                                                                           4    120  1000    28     5                                           gium vulgare                                                     AT 10 ATCC 19190                                                                             Streptosporan-                                                                           1    120  1000    28     4                                           gium india-                                                                    nensis                                                           AT 10 ATCC 19190                                                                             Streptosporan-                                                                           44   120  1000    28     5                                           gium india-                                                                    nensis                                                           SE 103                                                                               CBS 616.71                                                                             Actinoplanes                                                                             1    120  1000    28     4                                           spec.                                                            HN 6  CBS 602.71                                                                             Actinobifida                                                                             44   120  1000    50     4                                           chromogena                                                       HN 2  CBS 603.71                                                                             Actinobifida                                                                             44   120  1000    50     5                                           chromogena                                                       AT 2  CBS 367.66                                                                             Actinoplanes                                                                             4    120  1000    28     4                                           utahensis                                                        SE 101                                                                               CBS 621.71                                                                             Planomonspora                                                                            1    120  1000    28     6                                           parontospora                                                     SK 2  CBS 620.71                                                                             Pilimelia spec.                                                                          1    120  1000    28     7                             SE 82 CBS 615.71                                                                             Actinoplanes                                                                             4    120  1000    28     5                                           spec.     1    120  1000    28     5                             SE 45 CBS 618.71                                                                             Ampullariella                                                                            4    120  1000    28     4                                           spec.                                                            SA 28 CBS 617.71                                                                             Ampullariella                                                                            1    120  1000    28     5                                           digitata                                                         SA 8  CBS 611.71                                                                             Actinoplanes                                                                             4    120  1000    28     5                                           spec.                                                            AT 7  ATCC 12428                                                                             Streptosporan-                                                   4     120     1000      28   4                                                               gium roseum                                                      __________________________________________________________________________     Strain Number      Activity of the Culture Filtrate U/ml                       Laboratory                                                                           Collection                                                                             Strain Name                                                                              Amylase Inhibition                                                                        Maltase Inhibition                                                                        Saccharase Inhibition            Reference                                                                              No.                                                                    __________________________________________________________________________     AT 4  CBS 191.64                                                                             Ampullariella        0.003      0.5                                            digitata                                                         AT 9  KCC-A 0028                                                                             Streptosporan-                  0.4                                            gium vulgare                                                     AT 10 ATCC 19190                                                                             Streptosporan-                  0.5                                            gium india-                                                                    nensis                                                           AT 10 ATCC 19190                                                                             Streptosporan-                  0.3                                            gium india-                                                                    nensis                                                           SE 103                                                                               CBS 616.71                                                                             Actinoplanes                                                                   spec.                                                            HN 6  CBS 602.71                                                                             Actinobifida                                                                   chromogena                                                       HN 2  CBS 603.71                                                                             Actinobifida                                                                   chromogena                                                       AT 2. CBS 367.66                                                                     Actinoplanes      0.003                                                                utahensis                                                        SE 101                                                                               CBS 621.71                                                                             Planomonospora                                                                 parontospora                                                     SK 2  CBS 620.71                                                                             Pilimeliaspec.                                                   SE 82 CBS 615.71                                                                             Actinoplanes                                                                              200       0.010      0.8                                            spec.     3000       0.008      0.8                              SE 45 CBS 618.71                                                                             Ampullariella                                                                  spec.                                                            SA 28 CBS 617.71                                                                             Ampullariella        0.002                                                     digitata                                                         SA 8  CBS 611.71                                                                             Actinoplanes                                                                   spec.                                                            AT 7  ATCC 12428                                                                             Streptosporan-                                                                 gium roseum                                                      __________________________________________________________________________     Strain Number      Activity of Mycelium Extract According to Example 21                           (U/ml)                                                      Laboratory                                                                           Collection                                                                             Strain Name                                                                              Amylase Inhibition                                                                        Maltase Inhibition                                                                        Saccharase Inhibition            Reference                                                                              No.                                                                    __________________________________________________________________________     AT 4  CBS 191.64                                                                             Ampullariella                                                                  digitata                                                         AT 9  KCC-A 0028                                                                             Streptosporan-       0.004      1.5                                            gium vulgare                                                     AT 10 ATCC 19190                                                                             Streptosporan-                                                                 gium india-                                                                    nensis                                                           AT 10 ATCC 19190                                                                             Streptosporan-                  0.3                                            gium india-                                                                    nensis                                                           SE 103                                                                               CBS 616.71                                                                             Actinoplanes                    0.5                                            spec.                                                            HN 6  CBS 602.71                                                                             Actinobifida                    1.5                                            chromogena                                                       HN 2  CBS 603.71                                                                             Actinobifida                    3.0                                            chromogena                                                       AT 2  CBS 367.66                                                                             Actinoplanes                                                                   utahensis                                                        SE 101                                                                               CBS 621.71                                                                             Planomonospora       0.006                                                     parontospora                                                     SK 2  CBS 620.71                                                                             Pilimeliaspec.                                                   SE 82 CBS 615.71                                                                             Actinoplanes                                                                             200        0.010      0.6                                            spec.     35000      0.003      0.6                              SE 45 CBS 618.71                                                                             Ampullariella                   0.2                                            spec.                                                            SA 28 CBS 617.71                                                                             Ampullariella        0.003                                                     digitata                                                         SA 8  CBS 611.71                                                                             Actinoplanes                                                                             200                                                                  spec.                                                            AT 7  ATCC 12428                                                                             Streptosporan-                                                                 gium roseum                                                      __________________________________________________________________________ 

What is claimed is:
 1. A method for the production of an inhibitor of glycosidehydrolase enzymes of the digestive tract comprising culturing a microorganism of the order Actinomycetales and extracting the inhibitor from the resulting culture, wherein said inhibitor:a. is substantially heat-stable at neutral pH values; b. is stable to acid at pH 2; c. is stable to alkali at pH 12; d. is slowly dialyzable; e. is not inactivated by trypsin or pepsin; f. does not inhibit trypsin or pepsin; g. is not receptive to protein dyes; h. is free from characteristic UV absorption up to 250 nm.; i. is not inhibited by urea or β-mercaptoethanol; j. is of molecular weight 500-6000; and k. yields a monosaccharide on hydrolytic splitting.
 2. The method of claim 1 in which the microorganism is of the family Streptomycetaceae.
 3. The method of claim 1 in which the microorganism is of the family Actinoplanaceae.
 4. The method of claim 1 in which the microorganism is a member of the genera Actinoplanes, Ampullariella, Streptosporangia, Streptomyces, Chainia, Pilimelia, Planomonospora, or Actinobifida.
 5. The method of claim 1 in which the inhibitor is extracted from the culture broth.
 6. The method of claim 5 in which the inhibitor is extracted by concentration of the broth followed by filtration or centrifugation to remove insoluble material.
 7. The method of claim 5 in which the inhibitor is extracted from the broth, optionally after concentration, by precipitation with a water-soluble organic solvent.
 8. The method of claim 5 in which the inhibitor is extracted from the broth, optionally after concentration, by salting out.
 9. The method of claim 5 in which the inhibitor is extracted from the broth, optionally after concentration, by adsorption on and elution from an ion exchange resin.
 10. The method of any preceding claim in which the inhibitor is extracted from the mycelium.
 11. The method of claim 10 in which the inhibitor is extracted with an organic solvent.
 12. The method of claim 10 in which the inhibitor is extracted with acetone and then with ether.
 13. The method of claim 11 in which the mycelium is then dried and extracted with dimethyl sulfoxide. 